Methods of stimulating protective immunity employing Dengue viral antigens

ABSTRACT

Compositions that include at least a portion of at least one pathogen-associated molecular pattern and at least a portion of at least one member selected from the group consisting of a Den1 viral envelope protein, a Den2 viral envelope protein, a Den3 viral envelope protein and a Den4 viral envelope protein are employed in methods to stimulate a protective immune response in a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/050,938, filed Oct. 10, 2013, which is a divisional of U.S.application Ser. No. 11/879,695, filed Jul. 18, 2007, now U.S. Pat. No.8,574,588, which is a continuation-in-part application of InternationalApplication No. PCT/US2006/001623, which designated the United Statesand was filed on Jan. 19, 2006, published in English, which claims thebenefit of U.S. Provisional Application Nos. 60/645,170, filed Jan. 19,2005; 60/653,405, filed Feb. 15, 2005; 60/704,160, filed Jul. 29, 2005;60/723,409, filed Oct. 4, 2005; and 60/725,919, filed Oct. 11, 2005. Theteachings of the above applications are incorporated herein by referencein their entirety.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

a) File name: 37101010023SEQLIST.txt; created Sep. 19, 2014, 252 KB insize.

BACKGROUND OF THE INVENTION

Infections with viruses, including flaviviruses, such as West Nileflavivirus, Dengue flavivirus, Japanese encephalitis flavivirus, Langatflavivirus, Kunjin flavivirus, Murray Valley encephalitis flavivirus,Tick-borne flavivirus and Yellow fever flavivirus, can result in seriousdisease and, possibly death. Mosquitoes and ticks transmit many of theflaviviruses. For example, severe symptoms of West Nile virus infectioninclude high fever, headache, neck stiffness, stupor, disorientation,coma, tremors, convulsions, muscle weakness, vision loss, numbness,meningoencephalitis and paralysis. These symptoms may last severalweeks, and neurological effects may be permanent. In cases with mildersymptoms (e.g., fever, headache, and body aches, nausea, vomiting, andsometimes swollen lymph glands or a skin rash on the chest, stomach andback), certain symptoms, such as fever and aches, can pass on their own.In more severe cases, people usually require hospitalization fortreatment, such as administration of intravenous fluids and assistancewith breathing.

Methods to prevent flavivirus infection include compositions of liveattenuated and inactivated virus. However, such compositions may be lessthan optimally immunogenic, may result in unknown hazards if improperlyprepared and may have adverse side effects. There is a need to developnew compositions and methods to prevent flavivirus infection.

SUMMARY OF THE INVENTION

The present invention relates to compositions, fusion proteins andpolypeptides of at least a portion of an antigen and a flagellin thatlacks a hinge region; and at least a portion of at least onepathogen-associated molecular pattern (PAMP) and at least a portion ofat least one flavivirus. The compositions, fusion protein andpolypeptides of the invention can be employed in methods to stimulate animmune response and protective immunity in a subject.

In one embodiment, the invention is a composition comprising at least aportion of at least one antigen and at least a portion of at least oneflagellin, wherein at least one of the flagellin lacks at least aportion of a hinge region.

In another embodiment, the invention is a fusion protein comprising atleast a portion of at least one antigen and at least a portion of atleast one flagellin, wherein at least one of the flagellin lacks atleast a portion of a hinge region.

In an additional embodiment, the invention is a composition comprisingat least a portion of at least one pathogen-associated molecular patternand at least a portion of at least one viral protein selected from thegroup consisting of a West Nile viral protein, a Langat viral protein, aKunjin viral protein, a Murray Valley encephalitis viral protein, aJapanese encephalitis viral protein, a tickborne encephalitis viralprotein, and a Yellow fever viral protein.

In yet another embodiment, the invention is a composition comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one Den2 viral envelope protein,wherein the Den2 viral envelope protein is at least one member selectedfrom the group consisting of SEQ ID NO: 22 and SEQ ID NO: 40.

In another embodiment, the invention is a composition comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one member selected from the groupconsisting of a Den1 viral envelope protein, a Den2 viral envelopeprotein, a Den3 viral envelope protein and a Den4 viral envelopeprotein.

In still another embodiment, the invention is a fusion proteincomprising at least a portion of at least one pathogen-associatedmolecular pattern and at least a portion of at least one viral proteinselected from the group consisting of a West Nile viral protein, aLangat viral protein, a Kunjin viral protein, a Murray Valleyencephalitis viral protein, a Japanese encephalitis viral protein, atickborne encephalitis viral protein, and a Yellow fever viral protein.

An additional embodiment of the invention is a fusion protein comprisingat least a portion of at least one member selected from the groupconsisting of a Salmonella typhimurium flagellin type 2 (fljB/STF2), anE. coli fliC, and a S. muenchen fliC and at least a portion of at leastone member selected from the group consisting of a Den1 viral envelopeprotein, a Den2 viral envelope protein, a Den3 viral envelope proteinand a Den4 viral envelope protein.

In another embodiment, the invention is a fusion protein comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one member selected from the groupconsisting of a Den1 viral envelope protein, a Den2 viral envelopeprotein, a Den3 viral envelope protein and a Den4 viral envelopeprotein.

Another embodiment of the invention is a polypeptide encoded by SEQ IDNO: 29.

In yet another embodiment, the invention is a polypeptide that includesSEQ ID NO: 30.

In a further embodiment, the invention is a polypeptide having at leastabout 85% identity to SEQ ID NO: 30.

In still another embodiment, the invention is a polypeptide encoded bySEQ ID NO: 31.

In another embodiment, the invention is a polypeptide that includes SEQID NO: 32.

In an additional embodiment, the invention is a polypeptide having atleast about 70% identity to SEQ ID NO: 32.

In yet another embodiment, the invention is a polypeptide encoded by SEQID NO: 33.

In another embodiment, the invention is a polypeptide that includes SEQID NO: 34.

In still another embodiment, the invention is a polypeptide having atleast about 70% identity to SEQ ID NO: 34.

In an additional embodiment, the invention is a polypeptide encoded bySEQ ID NO: 35.

In a further embodiment, the invention is a polypeptide that includesSEQ ID NO: 36.

In yet another embodiment, the invention is a polypeptide having atleast 80% identity to SEQ ID NO: 36.

In another embodiment, the invention is a polypeptide encoded by SEQ IDNO: 37.

In still another embodiment, the invention is a polypeptide thatincludes SEQ ID NO: 38.

In another embodiment, the invention is a polypeptide having at least70% identity to SEQ ID NO: 38.

In an additional embodiment, the invention is a polypeptide encoded bySEQ ID NO: 54.

In another embodiment, the invention is a polypeptide that includes SEQID NO: 55.

Another embodiment of the invention is a polypeptide having at leastabout 70% identity to SEQ ID NO: 55.

In still another embodiment, the invention is a polypeptide thatincludes at least one member selected from the group consisting of SEQID NO: 71 and SEQ ID NO: 72.

In another embodiment, the invention is a polypeptide encoded by atleast one member selected from the group consisting of SEQ ID NO: 70 andSEQ ID NO: 73.

In yet another embodiment, the invention is a polypeptide having atleast about 70% identity to at least one member selected from the groupconsisting of SEQ ID NO: 71 and SEQ ID NO: 72.

In still another embodiment, the invention is a polypeptide thatincludes at least one member selected from the group consisting of SEQID NO: 76 and SEQ ID NO: 6.

In a further embodiment, the invention is a polypeptide encoded by atleast one member selected from the group consisting of SEQ ID NO: 77 andSEQ ID NO: 5.

In another embodiment, the invention is a polypeptide having at leastabout 70% identity to at least one member selected from the groupconsisting of SEQ ID NO: 76 and SEQ ID NO: 6.

In an additional embodiment, the invention is a polypeptide thatincludes at least one member selected from the group consisting of SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

In still another embodiment, the invention is a polypeptide encoded byat least one member selected from the group consisting of SEQ ID NO: 81,SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87.

In a further embodiment, the invention is a polypeptide having at leastabout 70% identity to at least one member selected from the groupconsisting of SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO:86.

In an additional embodiment, the invention is a polypeptide thatincludes SEQ ID NO: 159.

In yet another embodiment, the invention is a polypeptide encoded by SEQID NO: 158.

In another embodiment, the invention is a polypeptide having at leastabout 70% identity to SEQ ID NO: 159.

In yet another embodiment, the invention is a composition comprising atleast one Pam3Cys and at least a portion of at least one flavivirusprotein.

In an additional embodiment, the invention is a composition comprisingat least one Pam2Cys and at least a portion of at least one flavivirusprotein.

In still another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one viral protein selected from the group consisting of a WestNile viral protein, a Langat viral protein, a Kunjin viral protein, aMurray Valley encephalitis viral protein, a Japanese encephalitis viralprotein, a tickborne encephalitis viral protein, and a Yellow fevervirus protein.

In a further embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one Den2 envelope protein, wherein the Den2 envelope protein isselected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 40.

In yet another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a tickborne encephalitis viral protein and a Yellow feverviral protein.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one member selected from the group consisting of a Den1 viralenvelope protein, a Den2 viral envelope protein, a Den3 viral envelopeprotein and a Den4 viral envelope protein.

In still another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one member selected from the group consisting of a Salmonellatyphimurium flagellin type 2 (fljB/STF2), an E. coli fliC, and a S.muenchen fliC and at least a portion of at least one member selectedfrom the group consisting of a Den1 viral envelope protein, a Den2 viralenvelope protein, a Den3 viral envelope protein and a Den4 viralenvelope protein.

In an additional embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In a further embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition comprising at least a portion of at least oneantigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lack at least a portion of a hinge region.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein comprising at least a portion of at leastone antigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lack at least a portion of a hinge region.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a Tick-borne encephalitis viral protein, and a Yellowfever virus protein.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one Den2 envelope protein, wherein the Den2 envelope proteinis at least one member selected from the group consisting of SEQ ID NO:22, SEQ ID NO: 40 and SEQ ID NO: 97.

In still another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a Tick-borne encephalitis viral protein and a Yellowfever viral protein.

In an additional embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one member selected from the group consisting of a Salmonellatyphimurium flagellin type 2 (fljB/STF2), an E. coli fliC, and a S.muenchen fliC and at least a portion of at least one member selectedfrom the group consisting of a Den1 viral envelope protein, a Den2 viralenvelope protein, a Den3 viral envelope protein and a Den4 viralenvelope protein.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition comprising at least a portion of at leastone antigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lacks at least a portion of a hinge region.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein comprising at least a portion of atleast one antigen and at least a portion of at least one flagellin,wherein at least one of the flagellins lacks at least a portion of ahinge region.

The compositions, fusions proteins and polypeptides of the invention canbe employed to stimulate an immune response or protective immunity in asubject. Advantages of the claimed invention can include, for example,prevention of flavivirus infection in a subject in a manner specific fora particular antigen or virus, such as a flavivirus protein, that haseffective immunogencity and reduced side effects. The claimedcompositions, fusion proteins, polypeptides and methods can be employedto prevent or treat infection and, therefore, avoid serious diseasesconsequent to antigen or viral infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequence (SEQ ID NO: 1) of Salmonellatyphimurium flagellin type 2 (fljB/STF2, also referred to herein as“STF2”). The hinge region (also referred to herein as “thehypervariable” or the “hypervariable hinge region”) is underlined.

FIG. 2 depicts the nucleic acid sequence (SEQ ID NO: 2) encoding SEQ IDNO: 1. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 3 depicts the amino acid sequence (SEQ ID NO: 3) of a fljB/STF2Δ(also referred to herein as “fljB/STF2Δ” or “STF2Δ”). STF2Δ is a STF2lacking at least a portion of the hinge region. The artificial hingeregion is underlined.

FIG. 4 depicts the nucleic acid sequence (SEQ ID NO: 4) encoding SEQ IDNO: 3. The nucleic acid sequence encoding the artificial hinge region isunderlined.

FIG. 5 depicts the nucleic acid sequence (SEQ ID NO: 5) encoding apET/STF2Δ.JEIII+ fusion protein. The nucleic acid sequence encoding theartificial hinge region is double underlined. The nucleic acid sequenceencoding the linker between STF2Δ and JEIII+ is underlined. The nucleicacid sequence encoding JEIII+ is bolded.

FIG. 6 depicts the amino acid sequence (SEQ ID NO: 6) encoded by SEQ IDNO: 5. The artificial hinge is double underlined. The linker betweenSTF2Δ and JEIII+ is underlined. The amino acid sequence of JEIII+ isbolded.

FIG. 7 depicts the nucleic acid sequence (SEQ ID NO: 29) encoding aSTF2.EIII+ fusion protein. The nucleic acid sequence encoding the hingeregion of STF2 is underlined.

FIG. 8 depicts the amino acid sequence (SEQ ID NO: 30) encoded by SEQ IDNO: 29. The hinge region of STF2 is underlined.

FIG. 9 depicts the nucleic acid sequence (SEQ ID NO: 31) encoding aSTF2Δ.EIII+ fusion protein. The naturally occurring hinge region of STF2has been removed and replaced with an artificial hinge region. Thenucleic acid sequence encoding the artificial hinge region isunderlined. The nucleic acid sequence encoding EIII+ is bolded.

FIG. 10 depicts the amino acid sequence (SEQ ID NO: 32) encoded by SEQID NO: 31. The artificial hinge region is underlined. The EIII+ aminoacid sequence is bolded.

FIG. 11 depicts the nucleic acid sequence (SEQ ID NO: 33) of aSTF2Δ.EIII+ fusion protein. The nucleic acid sequence encoding theartificial hinge region is double underlined. The nucleic acid sequenceencoding a linker between STF2Δ and EIII+ is underlined. The nucleicacid sequence encoding EIII+ is bolded. Vector sequence is unbolded atthe 3′ end of the nucleic acid sequence.

FIG. 12 depicts the amino acid sequence (SEQ ID NO: 34) encoded by SEQID NO: 33. The artificial hinge region is double underlined. The linkerbetween STF2Δ and EIII+ is underlined. The amino acid sequence of theEIII+ is bolded. Domain I of the West Nile virus protein is bolded anditalicized (MEKLQ, SEQ ID NO: 172). The remainder of the bolded sequence(LKGTTYGVCSKAFKFLGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIGK, SEQ ID NO: 176) isdomain III of the envelope protein of the West Nile virus. Vectorsequence at the carboxy-terminus is not bolded at the carboxy-terminus.

FIG. 13 depicts the nucleic acid sequence (SEQ ID NO: 35) of aSTF2.EIII+ fusion protein. The nucleic acid sequence encoding the hingeregion of STF2 is underlined. The nucleic acid sequence encoding alinker between STF2 and EIII+ is bolded and underlined. The nucleic acidsequence encoding EIII+ is bolded.

FIG. 14 depicts the amino acid sequence (SEQ ID NO: 36) encoded by SEQID NO: 35. The hinge region is underlined. The linker between STF2 andEIII+ is bolded and underlined. The amino acid sequence of EIII+ isbolded.

FIG. 15 depicts the nucleic acid sequence (SEQ ID NO: 37) encoding afljB/STF2Δ.EIII+ fusion protein. There is no linker between STFΔ andEIII+.

FIG. 16 depicts the amino acid sequence (SEQ ID NO: 38) encoded by SEQID NO: 37. The amino acid sequence of EIII+ is bolded.

FIG. 17 depicts the nucleic acid sequence (SEQ ID NO: 54) of afljB/STF2.EIII+ fusion protein. The nucleic acid sequence encoding thehinge region of STF2 is underlined. The nucleic acid sequence encoding alinker between STF2 and EIII+ is bolded and underlined. The nucleic acidsequence encoding EIII+ is bolded.

FIG. 18 depicts the amino acid sequence (SEQ ID NO: 55) encoded by SEQID NO: 54. The amino acid sequence of the hinge region of STF2 isunderlined. The amino acid sequence of the linker between STF2 and EIII+is bolded and underlined. The amino acid sequence of EIII+ is bolded.

FIG. 19 depicts the amino acid sequence (SEQ ID NO: 58) of Salmonellamuenchen flagellin fliC. The amino acid sequence of the hinge region isunderlined.

FIG. 20 depicts the nucleic acid sequence (SEQ ID NO: 59) encoding SEQID NO: 58. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 21 depicts the nucleic acid sequence (SEQ ID NO: 63) of a linker.

FIG. 22 depicts the amino acid sequence (SEQ ID NO: 64) of Hepatitis CE1.

FIG. 23 depicts the amino acid sequence (SEQ ID NO: 65) of Hepatitis CE2.

FIG. 24 depicts the nucleic acid sequence (SEQ ID NO: 66) encoding SEQID NO: 64.

FIG. 25 depicts the nucleic acid sequence (SEQ ID NO: 67) encoding SEQID NO: 65.

FIG. 26 depicts the amino acid sequence (SEQ ID NO: 68) of E. Coli fliC.The amino acid sequence of the hinge region is underlined.

FIG. 27 depicts the nucleic acid sequence (SEQ ID NO: 69) encoding SEQID NO: 68. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 28 depicts the nucleic acid sequence (SEQ ID NO: 70) encoding afljB/STF2Δ.EIII+ fusion protein. The nucleic acid sequence encoding theartificial hinge region is double underlined. The nucleic acid sequenceencoding a linker between STF2Δ and EIII+ is underlined. The nucleicacid sequence encoding the EIII+ is bolded. Vector sequence is notbolded at the 3′ end of the sequence.

FIG. 29 depicts the amino acid sequence (SEQ ID NO: 71) encoded by SEQID NO: 70. The artificial hinge region is double underlined. The aminoacid sequence of the linker between STF2Δ and EIII+ is underlined. Theamino acid sequence of the EIII+ is bolded. Vector sequence at thecarboxy-terminus is not bolded.

FIG. 30 depicts the amino acid sequence (SEQ ID NO: 72) of afljB/STF2Δ.EIIIs+ fusion protein. The artificial hinge region is doubleunderlined. The amino acid sequence encoding the linker between STF2Δand EIII+ is underlined. Domain I of the West Nile virus protein isbolded and italicized (SEQ ID NO: 172). The remainder of the boldedsequence is domain III of the envelope protein (SEQ ID NO: 176) of theWest Nile virus. Portions of domains I and III are referred to as EIII+.Vector sequence at the carboxy-terminus of the protein is unbolded. Theserine residue of the linker region is bolded and is a substitution ofthe cysteine residue in the same region of the linker of SEQ ID NO: 71of FIG. 29.

FIG. 31 depicts the nucleic acid sequence (SEQ ID NO: 73) encoding SEQID NO: 72. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding thelinker between STF2Δ and EIII+ is underlined with the codon encoding theserine residue bolded. The nucleic acid sequence encoding EIII+ isindicated by bolded text. Linker sequence is unbolded text at the 3′end.

FIG. 32 depicts the amino acid sequence (SEQ ID NO: 76) of apET/STF2Δ.JEIII+ fusion protein. The artificial hinge region is doubleunderlined. The amino acid sequence of the linker between STF2Δ andJEIII+ is underlined. The amino acid sequence of a portion of domain Iof the Japanese encephalitis virus is bolded and italicized (MDKLAL, SEQID NO: 173). The amino acid sequence of a portion of the domain III ofthe Japanese encephalitis virus is bolded(KGTTYGMCTEKFSFAKNPVDTGHGTVVIELSYSGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLGKA, SEQ ID NO: 177).Portions of domains I and III are referred to as “JEIII+.”

FIG. 33 depicts the nucleic acid sequence (SEQ ID NO: 77) encoding SEQID NO: 76. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding a linkerbetween STF2Δ and JEIII+ is underlined. The nucleic acid sequenceencoding a portion of domain I of the Japanese encephalitis virus isbolded and italicized. The nucleic acid sequence encoding a portion ofdomain III of the Japanese encephalitis virus is bolded. Portions ofdomains I and III are referred to as “JEIII+.”

FIG. 34 depicts the nucleic acid sequence (SEQ ID NO: 78) encodingJEIII+. The nucleic acid sequence encoding at least a portion of domainI of the envelope protein is underlined. The remaining nucleic acidsequence encodes at least a portion of domain III of the envelopeprotein.

FIG. 35 depicts the amino acid sequence (SEQ ID NO: 79) encoded by SEQID NO: 78. At least a portion of domain I of the envelope protein isbolded and italicized. The remaining sequence is at least a portion ofdomain III of the envelope protein.

FIG. 36 depicts the amino acid sequence (SEQ ID NO: 80) of apET/STF2Δ.Den1 EIII fusion protein. The artificial hinge region isdouble underlined. A linker between STF2Δ and Den1 EIII is underlined.

FIG. 37 depicts the nucleic acid sequence (SEQ ID NO: 81) encoding SEQID NO: 80. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding thelinker between STF2Δ and Den1 EIII is underlined.

FIG. 38 depicts the amino acid sequence (SEQ ID NO: 82) of apET/STF2Δ.Den2 EIII fusion protein. The artificial hinge region isdouble underlined. The amino acid sequence of the linker between STF2Δand Den2 EIII is underlined.

FIG. 39 depicts the nucleic acid sequence (SEQ ID NO: 83) encoded by SEQID NO: 82. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding thelinker between STF2Δ and Den2 EIII is underlined.

FIG. 40 depicts the amino acid sequence (SEQ ID NO: 84) of apET/STF2Δ.Den3 EIII fusion protein. The artificial hinge region isdouble underlined. The amino acid sequence of the linker between STF2Δand Den3 EIII is underlined.

FIG. 41 depicts the nucleic acid sequence (SEQ ID NO: 85) encoding SEQID NO: 84. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding thelinker between STF2Δ and Den3 EIII is underlined.

FIG. 42 depicts the amino acid sequence (SEQ ID NO: 86) of apET/STF2Δ.Den4 EIII fusion protein. The artificial hinge region isdouble underlined. The amino acid sequence of the linker between STF2Δand Den4 EIII is underlined.

FIG. 43 depicts the nucleic acid sequence (SEQ ID NO: 87) encoding SEQID NO: 86. The nucleic acid sequence encoding the artificial hingeregion is double underlined. The nucleic acid sequence encoding thelinker between STF2Δ and Den4 EIII is underlined.

FIG. 44 depicts the amino acid sequence (SEQ ID NO: 174) of the envelopeprotein of the Tick-borne encephalitis envelope protein.

FIG. 45 depicts the amino acid sequence (SEQ ID NO: 39) of a West Nilevirus envelope protein (WNE) (amino acids 1-406). The amino acidsequence incorporated into EIII+ constructs is underlined (amino acids292-406). Amino acids 292-297 correspond to a portion of domain I; aminoacids 298-406 correspond to domain III. SEQ ID NO: 39 is encoded by SEQID NO: 57 (FIG. 67).

FIG. 46 depicts fusion constructs in a pET24 vector. T7:T7 promoter;lacO:lac operator; STF2: Salmonella typhimurium flagellin; STF2Δ=STF2with the hinge region deleted; EIII³⁰ is domain III of a West Nileenvelope protein with 6 amino acids of domain I amino acid.

FIGS. 47A and 47B depict TLR-5 bioactivity of STF2.EIII+ (SEQ ID NOS:54, 55) and STF2ΔEIII+ (SEQ ID NOS: 70, 71) fusion proteins. Serialdilutions of purified proteins were added to HEK293 (TLR5+) cellsovernight and IL-8 content of the supernatants measured by ELISA.Purified STF2.OVA was used as a positive control (FIG. 47A). The TLR-2agonist Pam3CSK4 was used as a negative control (FIG. 47B).

FIG. 48 depicts STF2Δ.EIII+ antigenic epitopes assessed by ELISA. Plateswere coated with full-length WNE (open bars) (SEQ ID NO: 39) orSTF2Δ.EIII+ (SEQ ID NOS: 70, 71) and probed with the indicatedantibodies (mAb). Poly=polyclonal antiserum to WNE; 3D9 through7H2=neutralizing monoclonal antibodies to WNE epitopes;anti-flagellin=monoclonal antibody to flagellin.

FIGS. 49A, 49B, 49C and 49D depict reactivity of STF2.E (SEQ ID NOS:158, 159); STF2.EIII+ (SEQ ID NOS: 54, 55) and STF2Δ.EIII+ (SEQ ID NOS:70, 71) fusion proteins with antibodies to WNE and flagellin. Plateswere coated with fusion proteins, blocked and incubated with antibodiesto WNE or flagellin. Antibody reactivity was detected followingincubation with HRP-labeled species specific IgG. Plates were developedin the presence of TMB substrate and O.D.450/650 using a TECAN platereader and Magellian software.

FIG. 50 depicts IgG serum following injection with fusion proteins. Micewere immunized with either PBS, Drosophila conditioned medium containingSTF2.E (CM, positive control), 25 μg of STF2Δ.EIII+ (SEQ ID NOS: 70, 71)i.p., 25 μg STF2Δ.EIII+ s.c., 25 μg STF2.EIII+ (SEQ ID NO: 54, 55) i.p.,25 μg STF2.EIII+ (SEQ ID NOS: 54, 55) or 25 μg STF2.E (SEQ ID NOS: 158,159). On day 35, immunized animals were challenged with WNV. Sera fromindividual mice (day 35) were characterized by direct ELISA to determineIgG levels. Purified WNV-E protein (SEQ ID NO: 39) was used as theantigen in this assay. This antigen (60) was produced in Drosophila as ahis-tagged protein.

FIG. 51 depicts STF2Δ.EIII+ (SEQ ID NOS: 70, 71) and STF2.EIII+ (SEQ IDNOS: 54, 55) protective immunity to WNV viral challenge. Mice wereimmunized and challenged with a lethal dose of WNV strain 2741 on day35. Survival was monitored for 21 days.

FIG. 52 depicts IgG sera titers following immunization with fusionproteins. STF2Δ.EIII+ proteins induce WNV-specific IgG antibodies. Micewere immunized s.c. on days 0, 14 and 28 with PBS alone or about 25 μgof STF2Δ.EIII+ (SEQ ID NOS: 70, 71) (045 [positive control]),STF2Δ.EIII+ (067, trimer), STF2Δ.EIII+ (070, monomer) orSTF2Δ.EIIIs+(SEQ ID NOS: 72, 73) (069). On day 35 sera from individualmice were characterized by direct ELISA to determine IgG levels.Purified WNV-E protein (060, produced in Drosophila as a his-taggedprotein) was used as the antigen in this assay.

FIG. 53 depicts STF2Δ.EIII+ (SEQ ID NOS: 70, 71) and STF2Δ.EIIIs+(SEQ IDNOS: 72, 73) protective immunity in mice from WNV lethal challenge. Onday 38 following immunization with fusion proteins, all groups werechallenged with a lethal dose of WNV strain 2741 and survival wasmonitored for 21 days. Survival for each group (10 mice/group) isindicated as a percentage.

FIG. 54 depicts competition assays. Serial dilutions (five fold startingat 1:25) of antisera from immunized animals were incubated withbiotinylated WNE protein (SEQ ID NO: 39) and then added to the wells ofELISA plates coated with mAb 7H2 at about 2 mg/ml. Wells were developedusing avidin-HRP to determine inhibition of West Nile protein binding asa results of competition with mAb 7H2.

FIG. 55 depicts epitope mapping of the antibody response induced bySTF2Δ.EIII+ (SEQ ID NOS: 72, 73) fusion proteins. Immune sera fromanimals immunized with indicated STF2Δ-fusion proteins (E2-21, E27-E52,FIG. 60) were examined for the ability to recognize overlapping peptidescorresponding to the junction of domains I and III of the WNV envelopeprotein.

FIG. 56 depicts epitope mapping of the antibody response induced bySTFΔ.EIIIs+ (SEQ ID NOS: 72, 73) E-21 (envelope protein) epitope fusionproteins. Immune sera from animals immunized with the indicatedSTF2Δ-fusion proteins (E2-21, E2-21-1 (S,C), E2-21-2(C,S), E2-21-2(C,S)and E2-21-4 through E2-21-24, see FIG. 57) were evaluated to identifythe residues defining the E-21 epitope of West Nile envelope protein.Data reflects the response of sera to E-21 following the substitution ofcysteine with serine (indicated by C,S); and the sequential replacementof amino acids with alanine. The peptides tested are listed in FIG. 57.

FIG. 57 depicts EIII+ peptide arrays. The sequences include domains Iand III of the West Nile virus envelope protein. Amino acids thatcorrespond to domain III are underlined. Amino acids that are notunderlined correspond to domain I.

FIG. 58 depicts Pam3Cys.WNV001 (SEQ ID NO: 168) inducing EIII specificIgG antibodies. Mice were immunized s.c. on days 0, 14 and 28 with PBSalone, 22 mg of unmodified WNV001 (SEQ ID NO: 168) or 30 μg ofPam3Cys.WNV001. On day 35 sera from individual mice were characterizedby direct ELISA to determine IgG levels to synthetic WNV001 peptide.

FIG. 59 depicts the amino acid sequences (SEQ ID NOS: 88-95) of theE1/EIII junction for West Nile, Japanese encephalitis and Dengue(serotypes 1 through 4) viruses. The West Nile epitope identified usingantisera from STF2Δ.EIIIs+ immunized animals is underlined. Thissequence corresponds to peptide E2-21 (SEQ ID NO: 125).

FIG. 60 depicts E2-21 peptide (SEQ ID NOS: 125-151) alanine scan array.Amino acids that correspond to domain III of the West Nile virusenvelope protein are underlined. Amino acids that are not underlinedcorrespond to domain I of the West Nile virus.

FIG. 61 depicts a STF2.OVA nucleic acid sequence (SEQ ID NO: 152). Thenucleic acid sequence encoding the linker between STF2 and ovalbumin(OVA) is underlined. Vector sequence at the 3′ end is bolded andunderlined.

FIG. 62 depicts an amino acid sequence (SEQ ID NO: 153) encoded by SEQID NO: 152. The linker sequence between STF2 and OVA is underlined.Vector sequence is underlined and bolded.

FIG. 63 depicts the amino acid sequence (SEQ ID NO: 154) of ovalbumin.

FIG. 64 depicts the nucleic acid sequence (SEQ ID NO: 155) of ovalbumin.

FIG. 65 depicts the nucleic acid sequence (SEQ ID NO: 158) encoding aSTF2.E fusion protein. The nucleic acid sequence encoding thefull-length West Nile virus envelope protein (E) is underlined.

FIG. 66 depicts the amino acid sequence (SEQ ID NO: 159) encoded by SEQID NO: 158. The amino acid sequence of the West Nile virus envelopeprotein is underlined.

FIG. 67 depicts the nucleic acid sequence (SEQ ID NO: 57) encoding SEQID NO: 39 (FIG. 45). The full length sequence of the West Nile virusenvelope protein is depicted.

FIG. 68 depicts the amino acid sequence (SEQ ID NO: 160) of the Dengue 1virus (also referred to herein as “Den-1,” “Den 1” or “Den1”).

FIG. 69 depicts the nucleic acid sequence (SEQ ID NO: 161) encoding SEQID NO: 160.

FIG. 70 depicts the amino acid sequence (SEQ ID NO: 162) of the Dengue 2virus (also referred to herein as “Den-2,” “Den 2” or “Den2”).

FIG. 71 depicts the nucleic acid sequence (SEQ ID NO: 163) encoding SEQID NO: 162.

FIG. 72 depicts the amino acid sequence (SEQ ID NO: 164) of the Dengue 3virus (also referred to herein as “Den-3,” “Den 3” or “Den3”).

FIG. 73 depicts the nucleic acid sequence (SEQ ID NO: 165) encoding SEQID NO: 164).

FIG. 74 depicts the amino acid sequence (SEQ ID NO: 166) of the Dengue 4virus (also referred to here in as “Den-4,” “Den 4” or “Den4”).

FIG. 75 depicts the nucleic acid sequence (SEQ ID NO: 167) encoding SEQID NO: 166.

FIG. 76 depicts the nucleic acid sequence (SEQ ID NO: 170) encoding aJapanese encephalitis virus.

FIG. 77 depicts the amino acid sequence (SEQ ID NO: 171) encoded by SEQID NO: 170.

FIG. 78 depicts the nucleic acid sequence (SEQ ID NO: 175) encoding SEQID NO: 174, depicted in FIG. 44.

FIG. 79 depicts the nucleic acid sequence (SEQ ID NO: 178) encodingEIII+ (amino acids of 292-406 of SEQ ID NO: 39, depicted in FIG. 45 andSEQ ID NO: 7).

FIG. 80 depicts a tripalmitoylated peptide.

FIGS. 81A and 81B depict anti-flagellin and anti-WNV-E specific IgGresponses in mice. Five groups of C3H/HeN mice (10 mice per group) wereimmunized on days 0, 14 and 28 days s.c. with STF2Δ.EIII (SEQ ID NO: 72;25 μg), STF2Δ (18 μg), WNV-EIIIs+(7 μg), and a mixture of STF2Δ (18 μg)and WNV-EIIIs+ (7 μg). Doses were chosen to ensure that molarequivalents of each antigen were administered in PBS. On day 35, serawere harvested and tested by ELISA for flagellin (81A) and WNV-E(81B)-specific IgG responses. Purified flagellin (STF2) and 80% WNE-Eprotein were used as antigens for antibody detection. Results reflectthe mean±standard error OD₄₅₀ values obtained from 10 individual animalsper group.

FIG. 82 depicts percent survival of immunized mice depicted in FIGS. 81Aand 81B challenged with a lethal dose (LD₉₀) of WNV-strain 2741 andmonitored for survival for 21 days.

FIGS. 83A and 83B depict IgG responses following immunization of wildtype or TLR5 knockout (ko) C57BL/6 mice with the STF2Δ.EIII+ fusionprotein (SEQ ID NO: 72). Wild type and TLR5ko mice (5 mice per group)were immunized with PBS, or 25 μg of the STF2Δ.EIII+ fusion protein s.c.on days 0 and 21, and sera were collected on day 28. Anti-flagellin andanti-E IgG responses were examined by ELISA. The data depict themean±standard deviation of 5 individual sera per group.

FIGS. 84A, 84B and 84C depict immunogenicity of STF2Δ.JEIII+ (SEQ ID NO:76) in C57BL/6 mice. Mice (20 mice per group) were immunized with PBS,2.5 μg of STF2Δ.JEIIIs+(SEQ ID NO: 76) on days 0, 14, or 28 and bled onday 7 (primary), day 21 (boost 1), and day 35 (boost 2). Anti-JE-his IgGresponses were examined by ELISA. The data depict the mean±SD of 20individual sera per group.

FIG. 85 depicts the percent survival of mice depicted in FIGS. 84A, 84Band 84C. Following the third immunization, 10 mice from each group werechallenged with of the Nakayama JE virus by i.p. administration with aviral dose of 10×LD₅₀. Survival was monitored for 21 days.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as a combination of parts of the invention, will now bemore particularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The present invention relates to compositions, fusion proteins andpolypeptides of at least a portion of at least one antigen and at leasta portion of a flagellin that lacks a hinge region; and at least aportion of at least one pathogen-associated molecular pattern (PAMP) andat least a portion of at least one flavivirus. The compositions, fusionproteins and polypeptides of the invention can be employed in methods tostimulate an immune response and protective immunity in a subject.

In one embodiment, the invention is a composition comprising at least aportion of at least one antigen and at least a portion of at least oneflagellin, wherein at least one of the flagellins lacks at least aportion of a hinge region.

Pathogen-associated molecular pattern (PAMP), such as a flagellin or abacterial lipoprotein, refers to a class of molecules (e.g., protein,peptide, carbohydrate, lipid, lipopeptide, nucleic acid) found inmicroorganisms that, when bound to a pattern recognition receptor (PRR),can trigger an innate immune response. The PRR can be a Toll-likereceptor (TLR). Toll-like receptors refer to a family of receptorproteins that are homologous to the Drosophila melangogaster Tollprotein. Toll-like receptors are type I transmembrane signaling receptorproteins characterized by an extracellular leucine-rich repeat domainand an intracellular domain homologous to an interleukin 1 receptor.Toll-like receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR 8, TLR9, TLR10, TLR11 and TLR12.

The pathogen-associated molecular pattern can be an agonist of atoll-like receptor, for example, a TLR2 agonist (i.e., Pam2Cys, Pam3Cys,a bacterial lipoprotein) or a TLR5 agonist, such as a flagellin.“Agonist,” as used herein in referring to a TLR, means a molecule thatactivates a TLR signaling pathway. A TLR signaling pathway is anintracellular signal transduction pathway employed by a particular TLRthat can be activated by a TLR ligand or a TLR agonist. Commonintracellular pathways are employed by TLRs and include, for example,NF-κB, Jun N-terminal kinase and mitogen-activated protein kinase. Thepathogen-associated molecular pattern can include at least one memberselected from the group consisting of a TLR1 agonist, a TLR2 agonist(e.g., Pam3Cys, Pam2Cys, bacterial lipoprotein), a TLR3 agonist (e.g.,dsRNA), a TLR4 agonist (e.g., bacterial lipopolysaccharide), a TLR5agonist (e.g., a flagellin), a TLR6 agonist, a TLR7 agonist, a TLR8agonist, a TLR9 agonist (e.g., unmethylated DNA motifs), TLR10 agonist,a TLR11 agonist and a TLR12 agonist.

TLR4 ligands (e.g., TLR4 agonists) for use in the compositions andmethods of the invention can include at least one member selected fromthe group consisting of SEQ ID NOS: 184-231 (see, PCT/US 2006/002906/WO2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865;PCT/US 2006/042051; U.S. application Ser. No. 11/714,873).

(SEQ ID NO: 184) GGKSGRTG (SEQ ID NO: 185) KGYDWLVVG  (SEQ ID NO: 186)EDMVYRIGVP (SEQ ID NO: 187) VKLSGS (SEQ ID NO: 188) GMLSLALF(SEQ ID NO: 189 CVVGSVR (SEQ ID NO: 190) IVRGCLGW (SEQ ID NO: 191)AAEERTLG (SEQ ID NO: 192) WARVVGWLR  (SEQ ID NO: 193) SEGYRLFGG (SEQ ID NO: 194) LVGGVVRRGS (SEQ ID NO: 195) GRVNDLWLAA (SEQ ID NO: 196)SGWMLWREGS (SEQ ID NO: 197) ERMEDRGGDL  (SEQ ID NO: 198) KLCCFTECM (SEQ ID NO: 199) AVGSMERGRG (SEQ ID NO: 200) RDWVGGDLV  (SEQ ID NO: 201)FFEVAKISQQ (SEQ ID NO: 202) WWYWC (SEQ ID NO: 203) MHLCSHA(SEQ ID NO: 204) WLFRRIG (SEQ ID NO: 205) YWFWRIG (SEQ ID NO: 206)MHLYCIA (SEQ ID NO: 207) WPLFPWIV (SEQ ID NO: 208) DMRSHAR(SEQ ID NO: 209) MHLCTHA (SEQ ID NO: 210) NLFPFY (SEQ ID NO: 211)MHLCTRA (SEQ ID NO: 212) RHLWYHA (SEQ ID NO: 213) WPFSAYW(SEQ ID NO: 214) WYLRGS (SEQ ID NO: 215) GKGTDLG (SEQ ID NO: 216) IFVRMR(SEQ ID NO: 217) WLFRPVF (SEQ ID NO: 218) FLGWLMG (SEQ ID NO: 219)MHLWHHA (SEQ ID NO: 220) WWFPWKA (SEQ ID NO: 221) WYLPWLG(SEQ ID NO: 222) WPFPRTF (SEQ ID NO: 223) WPFPAYW (SEQ ID NO: 224)FLGLRWL (SEQ ID NO: 225) SRTDVGVLEV (SEQ ID NO: 226) REKVSRGDKG(SEQ ID NO: 227) DWDAVESEYM (SEQ ID NO: 228) VSSAQEVRVP (SEQ ID NO: 229)LTYGGLEALG (SEQ ID NO: 230) VEEYSSSGVS (SEQ ID NO: 231) VCEVSDSVMA

TLR2 ligands (e.g., TLR2 agonists) for use in the compositions andmethods of the invention can also include at least one member selectedfrom the group consisting of SEQ ID NOS: 232-271 (see, PCT/US2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US2006/041865; PCT/US 2006/042051; U.S. application Ser. No. 11/714,873).

(SEQ ID NO: 232) NPPTT (SEQ ID NO: 233) MRRIL (SEQ ID NO: 234) MISS (SEQ ID NO: 235) RGGSK (SEQ ID NO: 236) RGGF  (SEQ ID NO: 237) NRTVF(SEQ ID NO: 238) NRFGL  (SEQ ID NO: 239) SRHGR  (SEQ ID NO: 240) IMRHP (SEQ ID NO: 241) EVCAP  (SEQ ID NO: 242) ACGVY  (SEQ ID NO: 243) CGPKL (SEQ ID NO: 244) AGCFS  (SEQ ID NO: 245) SGGLF  (SEQ ID NO: 246) AVRLS (SEQ ID NO: 247) GGKLS  (SEQ ID NO: 248) VSEGV  (SEQ ID NO: 249) KCQSF (SEQ ID NO: 250) FCGLG  (SEQ ID NO: 251) PESGV  (SEQ ID NO: 252) DPDSG (SEQ ID NO: 253) IGRFR  (SEQ ID NO: 254) MGTLP  (SEQ ID NO: 255) ADTHQ (SEQ ID NO: 256) HLLPG  (SEQ ID NO: 257) GPLLH  (SEQ ID NO: 258) NYRRW (SEQ ID NO: 259) LRQGR  (SEQ ID NO: 260) IMWFP  (SEQ ID NO: 261) RVVAP (SEQ ID NO: 262) IHVVP  (SEQ ID NO: 263) MFGVP  (SEQ ID NO: 264) CVWLQ (SEQ ID NO: 265) IYKLA  (SEQ ID NO: 266) KGWF  (SEQ ID NO: 267) KYMPH (SEQ ID NO: 268) VGKND  (SEQ ID NO: 269) THKPK (SEQ ID NO: 270) SHIAL(SEQ ID NO: 271) AWAGT

The TLR2 ligand (e.g., TLR2 agonist) can also include at least a portionof at least one member selected from the group consisting of flagellinmodification protein FlmB of Caulobacter crescentus; Bacterial Type IIIsecretion system protein; invasin protein of Salmonella; Type 4 fimbrialbiogenesis protein (PilX) of Pseudomonas; Salmonella SciJ protein;putative integral membrane protein of Streptomyces; membrane protein ofPseudomonas; adhesin of Bordetella pertusis; peptidase B of Vibriocholerae; virulence sensor protein of Bordetella; putative integralmembrane protein of Neisseria meningitidis; fusion of flagellarbiosynthesis proteins FliR and FlhB of Clostridium; outer membraneprotein (porin) of Acinetobacter; flagellar biosynthesis protein FlhF ofHelicobacter; ompA related protein of Xanthomonas; omp2a porin ofBrucella; putative porin/fimbrial assembly protein (LHrE) of Salmonella;wbdk of Salmonella; Glycosyltransferase involved in LPS biosynthesis;Salmonella putative permease.

The TLR2 ligand (e.g., TLR agonist) can include at least a portion of atleast one member selected from the group consisting oflipoprotein/lipopeptides (a variety of pathogens); peptidoglycan(Gram-positive bacteria); lipoteichoic acid (Gram-positive bacteria);lipoarabinomannan (mycobacteria); a phenol-soluble modulin(Staphylococcus epidermidis); glycoinositolphospholipids (TrypanosomaCruzi); glycolipids (Treponema maltophilum); porins (Neisseria); zymosan(fungi) and atypical LPS (Leptospira interrogans and Porphyromonasgingivalis).

The TLR2 ligand (e.g., TLR2 agonist) can also include at least onemember selected from the group consisting of SEQ ID NOS: 272-274 (see,PCT/US 2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792;PCT/US 2006/041865; PCT/US 2006/042051; U.S. application Ser. No.11/714,873).

(SEQ ID NO: 272) KGGVGPVRRSSRLRRTTQPG (SEQ ID NO: 273)GRRGLCRGCRTRGRIKQLQSAHK (SEQ ID NO: 274) RWGYHLRDRKYKGVRSHKGVPR 

In a particular embodiment, the TLR2 agonist is a bacterial lipoprotein,such as Pam2Cys, Pam3Cys or Pseudomonas aeruginosa OprI lipoprotein(OprI). Exemplary OprI lipoproteins include MNNVLKFSALALAAVLATGCSSH (SEQID NO: 179), encoded byATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCCAGCAAC (SEQ ID NO: 180). An exemplary E. coli bacteriallipoprotein for use in the invention described herein isMKATKLVLGAVILGSTLLAGCSSN (SEQ ID NO: 181) encoded byATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCCAGCAAC (SEQ ID NO: 182). A bacterial lipoprotein that activatesa TLR2 signaling pathway (a TLR2 agonist) is a bacterial protein thatincludes a palmitoleic acid (Omueti, K. O., et al., J. Biol. Chem. 280:36616-36625 (2005)). For example, expression of SEQ ID NOS: 180 and 182in bacterial expression systems (e.g., E. coli) results in the additionof a palmitoleic acid moiety to a cysteine residue of the resultingprotein (e.g., SEQ ID NOS: 179, 181) thereby generating a TLR2 agonistfor use in the compositions, fusion proteins and polypeptides of theinvention. Production of tripalmitoylated-lipoproteins (also referred toas triacyl-lipoproteins) in bacteria occurs through the addition of adiacylglycerol group to the sulfhydryl group of a cysteine (Cysteine 21of SEQ ID NO: 181) followed by cleavage of the signal sequence andaddition of a third acyl chain to the free N-terminal group of the samecysteine (Cysteine 21 of SEQ ID NO: 181) (Sankaran, K., et al., J. Biol.Chem. 269:19706 (1994)), to generate a tripalmitylated peptide (a TLR2agonist) as shown, for example, in FIG. 80.

An antigen is any molecule (e.g., protein, peptide, glycoprotein,glycopeptide, carbohydrate, lipid, lipopeptide, polysaccharide) thatgenerates an immune response in a subject either when employed incombination with a PAMP (e.g., a flagellin, Pam2Cys, Pam3Cys) or in theabsence of a PAMP. The antigen can be a fragment or portion of anaturally occurring antigen or a synthetic molecule that mimics thenaturally occurring antigen or a portion of the naturally occurringantigen.

The antigen can be a viral antigen. A “viral antigen,” as used herein,refers to any portion of a virus (e.g., flavivirus) that generates animmune response in a subject either when employed in combination with aPAMP (e.g., a flagellin, Pam2Cys, Pam3Cys) or in the absence of a PAMP.The viral antigen can be a portion or a fragment of a naturallyoccurring virus or a synthetic molecule that mimics a naturallyoccurring virus, such as a recombinant or synthetic protein (e.g., aflavivirus), peptide, lipid, carbohydrate, that generates an immuneresponse in the subject. “At least a portion,” as used herein inreference to at least a portion of an antigen (e.g., a viral antigen),means any part of the antigen or the entirety of the antigen. Forexample, at least a portion of a flaviviral antigen can be an envelopeprotein, or a domain (e.g., domain I, II, III) of an envelope protein ofa flavivirus antigen.

The flagellin employed in the compositions, fusion proteins andpolypeptides of the invention can lack at least a portion of a hingeregion. Hinge regions are the hypervariable regions of a flagellin thatlink the amino-terminus and carboxy-terminus of the flagellin. Hingeregions of a flagellin are also referred to herein as “hypervariableregions” or “hypervariable hinge regions.” “Lack,” as used herein inreference to a hinge region of a flagellin, means that at least oneamino acid or at least one nucleic acid codon encoding at least oneamino acid that comprises the hinge region of a flagellin is absent inthe flagellin. Example of hinge regions include amino acids 176-415 ofSEQ ID NO: 1, which are encoded by nucleic acids 528-1245 of SEQ ID NO:2; amino acids 174-422 of SEQ ID NO: 68, which are encoded by nucleicacids 522-1266 of SEQ ID NO: 69; or amino acids 173-464 of SEQ ID NO:58, which are encoded by nucleic acids 519-1392 of SEQ ID NO: 59. Thus,if amino acids 176-415 were absent from the flagellin of SEQ ID NO: 1,the flagellin would lack a hinge region. A flagellin lacking at least aportion of a hinge region is also referred to herein as a “truncatedversion” of a flagellin.

“At least a portion of a hinge region,” as used herein, refers to anypart of the hinge region of the PAMP (e.g., flagellin), or the entiretyof the hinge region. “At least a portion of a hinge region” is alsoreferred to herein as a “fragment of a hinge region.” For example, thehinge region of S. typhimurium flagellin B (fljB, also referred toherein as “fljB/STF2” or “STF2”) is amino acids 176-416 of SEQ ID NO: 1,which is encoded by nucleic acids at position 528-1245 of SEQ ID NO: 2.At least a portion of the hinge region of fljB/STF2 can be, for example,amino acids 200-300 of SEQ ID NO: 1. Thus, if amino acids 200-300 wereabsent from SEQ ID NO: 1, the resulting amino acid sequence of STF2would lack at least a portion of a hinge region.

At least a portion of a naturally occurring a flagellin can be replacedwith at least a portion of an artificial hinge region. “Naturallyoccurring,” as used herein in reference to a hinge region of aflagellin, means the hinge region that is present in the nativeflagellin. For example, amino acids 176-415 of SEQ ID NO: 1, amino acids174-422 of SEQ ID NO: 68 and amino acids 173-464 of SEQ ID NO: 58, arethe amino acids corresponding to the natural hinge region of STF2, E.coli fliC and S. muenchen flagellins, fliC, respectively. “Artificial,”as used herein in reference to a hinge region of a flagellin, means ahinge region that is inserted in the native flagellin in any region ofthe flagellin that contains or contained the native hinge region. Forexample, SEQ ID NO: 32 lacks the naturally occurring hinge region, whichhas been replaced by amino acids 176-186, the artificial hinge region.

An artificial hinge region may be employed in a flagellin that lacks atleast a portion of a hinge region to facilitate interaction of thecarboxy- and amino-terminus of the flagellin for binding to TLR5 and,thus, activation of the TLR5 innate signal transduction pathway. Aflagellin lacking at least a portion of a hinge region is designated bythe name of the flagellin followed by a “Δ.” For example, an STF2 (e.g.,SEQ ID NO: 1) that lacks at least a portion of a hinge region isreferenced to as “STF2Δ” or “fljB/STF2Δ” (e.g., SEQ ID NO: 3).

The flagellin employed in the compositions, fusion proteins andpolypeptides of the invention can be at least one member selected fromthe group consisting of fljB/STF2 (S. typhimurium flagellin B, GenbankAccession Number AF045151), a fragment of fljB/STF2, E. coli flagellinfliC (also referred to herein as E. coli fliC) (Genbank Accession NumberAB028476), a fragment of E. coli flagellin fliC, S. muenchen flagellinfliC (also referred to herein as S. muenchen fliC), and a fragment of S.muenchen flagellin fliC.

The flagellin employed in the compositions, fusion proteins andpolypeptides of the invention include the polypeptides of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 58 and SEQ ID NO: 68; at least a portion of SEQID NO: 1, at least a portion of SEQ ID NO: 3, at least a portion of SEQID NO: 58 and at least a portion of SEQ ID NO: 68; and a polypeptideencoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 59 and SEQ ID NO: 69;or at least a portion of a polypeptide encoded by SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 59 and SEQ ID NO: 69.

In another embodiment, the invention is a fusion protein comprising atleast a portion of at least one antigen and at least a portion of atleast one flagellin, wherein at least one of the flagellins lack atleast a portion of a hinge region.

“Fusion protein,” as used herein, refers to a protein generated from atleast two similar or distinct components (e.g., Pam2Cys, Pam3Cys, PAMP,at least a portion of an antigen, at least a portion of a viral protein)that are linked covalently or noncovalently. The components of thefusion protein can be made, for example, synthetically (e.g., Pam3Cys,Pam2Cys) or by recombinant nucleic acid techniques (e.g., transfectionof a host cell with a nucleic acid sequence encoding a component of thefusion protein, such as at least a portion of a PAMP, or at least aportion of an antigen or a viral protein). One component of the fusionprotein (e.g., Pam2Cys, Pam3Cys, PAMP, at least a portion of an antigenor at least a portion of a viral protein) can be linked to anothercomponent of the fusion protein (e.g., Pam2Cys, Pam3Cys, PAMP, at leasta portion of an antigen or at least a portion of a viral protein) usingchemical conjugation techniques, including peptide conjugation, or usingmolecular biological techniques, including recombinant technology, suchas the generation of a fusion protein construct. Chemical conjugation(also referred to herein as “chemical coupling”) can include conjugationby a reactive group, such as a thiol group (e.g., a cysteine residue) orby derivatization of a primary (e.g., a amino-terminal) or secondary(e.g., lysine) group. Exemplary fusion proteins of the invention includeSEQ ID NOS: 6, 71, 72, 76, 80, 82, 84, 86 and 159 (FIGS. 6, 29, 30, 32,36, 38, 40 and 42), encoded by SEQ ID NOS: 5, 70, 73, 77, 81, 83, 85, 87and 158 (FIGS. 5, 28, 31, 33, 37, 39, 41 and 43)

Fusion proteins of the invention can be designated by components of thefusion proteins separated by a “.” or “-.” For example, “STF2.EIII”refers to a fusion protein comprising one fljB/STF2 protein and at leasta portion of domain III (see, infra) of at least one West Nile virusenvelope protein; and “STF2Δ.EIII” refers to a fusion protein comprisingone fljB/STF2 protein lacking at least a portion of its hinge region andhaving at least a portion of domain III of at least one West Nile virusenvelope protein.

In yet another embodiment, the invention is a composition comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one viral protein selected from thegroup consisting of a West Nile viral protein, a Langat viral protein, aKunjin viral protein, a Murray Valley encephalitis viral protein, aJapanese encephalitis viral protein, a Tick-borne encephalitis viralprotein, and a Yellow fever viral protein, which are flaviviralproteins. The pathogen-associated molecular pattern and viral proteincan be components of a fusion protein.

The genus flavivirus is in the virus family Flaviviridae and consists ofabout 70 viruses. Mosquito or ticks transmit most of these viruses.Several flaviviruses are significant human pathogens, including the fourdengue viruses (Den1, Den2, Den3 and Den4), yellow fever (YF), Japaneseencephalitis (JE), West Nile (WN, also referred to herein as “WNV”) andTick-borne encephalitis (TBE) (Weaver S. C., et al., Nat Rev Microbiol10: 789-801 (2004)). The flavivirus genus is divided into a number ofserogroups based on cross-neutralization tests, including the dengueserogroup that contains four serologically and genetically distinctviruses termed DEN-1, DEN-2, DEN-3 and DEN-4.

Flaviviruses are small, enveloped viruses with icosahedral capsids. Theflavivirus genome is a single-stranded positive-sense RNA (about 11 kb)that is directly translated by the host cell machinery followinginfection. The viral genome is translated as a single polypeptide thatundergoes co- and post-translational cleavage by viral and cellularenzymes to generate three structural proteins of the flavivirus (thecapsid (C), the membrane (M) and the envelope (E) proteins); and sevennonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)(Weaver, et al., Annu Rev Microbiol 1990:44-649 (2004)). The flavivirusgenome organization is depicted in FIG. 44. The viral capsid is composedof the C-protein, while both the M- and envelope proteins are located onthe envelope surface of the virion (Weaver, S. C., et al., Nat. Rev.Microbiol. 10:789-801 (2004); Chambers et al., Annu Rev. Microbiol. 44:649-688 (1990)). A major immunogen for flaviviruses is the membraneenvelope protein.

The flavivirus envelope protein plays a role in virus assembly. Theseproteins form a protective shell around the virus, which serves as acage for the genetic material inside, sheltering the virus until it isreleased inside a host cell. While simple viruses consist of only aprotein shell and genetic information, more complex viruses, such asflaviviruses, also contain a lipid bilayer between the protein shell andviral genome. A flavivirus can enter a host cell when the viral envelopeprotein binds to a receptor and responds by conformational rearrangementto the reduced pH of an endosome. The conformational change inducesfusion of viral and host-cell membranes.

The envelope of a flavivirus may function as a receptor binding proteinand to facilitate fusion of the virus and host cell membrane. As areceptor binding protein, the envelope protein is a determinant of hostrange, cell tropism, virulence and elicits neutralizing antibodiesduring the immune response (Roehrig, Adv Virus Res 59:141-175 (2003)).The envelope protein is responsible for fusing the virus and hostmembranes (Chu, et al., J. Virol 78:10543-10555 (2004); Heinz, et al.,Adv Virus Res 59:63-97 (2003); Chu, et al., J. Gen Virol 86:405-412(2005)). Crystallographic structures of the Tick-borne encephalitisvirus envelope protein and the Dengue-2 (Den 2) virus envelope proteinhave been determined (Rey, et al., Nature 375:291-298 (1995); Modis, etal., Proc Natl Acad Sci USA 100:6986-6991 (2003)). Envelope proteins offlaviviruses have common structural (domains I, II and III) andfunctional features (receptor binding of virus and host cell and fusionfunctions) and are class II fusion glycoproteins (Lescar et al., Cell105:137-148 (2001)).

In the pre-fusion conformation, envelope proteins form homodimers on theouter surface of the virus particles (Rey, et al., Nature 375:291-298);Kuhn, et al., Cell 108:717-725 (2002); Mukhopadhyay, et al., Science302:248 (2003)). Each envelope protein monomer folds into threestructural domains (domains I, II and III) predominantly composed ofβ-strands. Domain I (also referred to herein as “I” or “DI”) iscentrally located in the structure and has an N-glycosylation site inglycosylated envelope proteins. Domain II (also referred to herein as“II” or “DII”) of the envelope protein promotes dimerization and has afusion loop that inserts into the target host membrane during thepH-dependent fusion of the virus (Modis, et al., Nature 427:313-319(2004); Bressanelli, et al., EMBO J 23:728-738 (2004)). Domain III (alsoreferred to herein as “III” or “DIII”) is at the carboxy-terminus of theenvelope protein. Domain III is also referred to as “domain B” inearlier antigenic mapping studies. Domain III has several epitopes thatcan elicit virus-neutralizing antibodies (Roehrig, Adv Virus Res59:141-175 (2003)). In addition, studies with several flaviviruses,including Tick-borne encephalitis (Mandle, et al., J. Virol 75:5627-5637(2001)), indicate that domain III, which has a fold typical of animmunoglobulin constant domain, may mediate flavivirus attachment tohost cells (Anderson, Adv Virus Res 59:229-274 (2003)) and, thus, be areceptor-binding domain.

The crystal structure of domains I, II and III of the envelope proteinfrom the Tick-borne encephalitis flavivirus and the Dengue 2 flavivirushas been determined (Rey, F. A., et al., Nature 375:291-298 (1995);Modis, Y., et al., Nature 427:313-319 (2004), respectively). Domain I ofthe Tick-borne encephalitis envelope protein corresponds to amino acids1-51, 137-189 and 285-302 of SEQ ID NO: 174; domain II of the Tick-borneencephalitis envelope protein of SEQ ID NO: 174 corresponds to aminoacids 52-136 and 190-284; and domain III corresponds to amino acids303-395 of SEQ ID NO: 174. (Rey, F. A., et al., Nature 375:291-298(1995)). SEQ ID NO: 174 (FIG. 44) is encoded by SEQ ID NO: 175 (FIG.78). Domain I of the Dengue 2 flavivirus envelope protein corresponds toamino acids 1-52, 132-193 and 280-296 of SEQ ID NO: 160 (FIG. 70);domain II corresponds to amino acids 53-131 and 194-279 of SEQ ID NO:160; and domain III corresponds to amino acids 297-495 of SEQ ID NO: 160(Modis, Y., et al., Nature 427:313-319 (2004)). The location of domainsI, II and III of other flavivirus (e.g., West Nile virus, Japaneseencephalitis, Dengue 1 virus, Dengue 3 virus and Dengue 4 virus) isbased on homology of the Tick-borne encephalitis envelope proteindomains and the Dengue 2 envelope protein domains. Thus, referenceherein to domains of flavivirus proteins, in particular, flavivirusesother than Tick-borne encephalitis flavivirus envelope proteins andDengue 2 flavivirus envelope proteins, are based on homology to domainsin the Tick-borne encephalitis flavivirus envelope protein and theDengue 2 flavivirus envelope protein.

The domain III of the envelope protein of the DEN flavivirus encodes themajority of the flavivirus type-specific contiguous critical/dominantneutralizing epitopes (Roehring, J. T., Adv. Virus Res. 59:141 (2003)),including the four DEN (DEN1, DEN2, DEN3, DEN4) viruses. Flavivirusenvelope proteins are highly homologous. Exemplary envelope proteinsequences are shown in FIGS. 45, 68, 70, 72, 74 and 77 (SEQ ID NOs: 39,160, 162, 164, 166 and 171, respectively).

The seven nonstructural proteins of flavivirus envelope proteins areinvolved in the replication of the virus. NS3 is a multifunctionalenzyme that encodes a serine protease at the a minus-terminal region;and helicase, RNA triphosphatase and NTPase activities in thecarboxy-terminal region. NS5 encodes a methyltransferase and theRNA-dependent-RNA polymerase. NS2A, NS2B, NS4A and NS4B are four poorlycharacterized proteins. The central domain of NS2B is a co-factor forthe NS3 serine protease while NS2A and NS4A are known to be componentsof the replication complex. NS1 is located at both the plasma membraneand in the lumen of intracellular vesicles of virus-infected cells. NS1is a multifunctional protein that is associated with an early step inthe replication cycle either prior to or early in negative-strand RNAsynthesis and is also thought to be involved in virus maturation and/orrelease (Brinton, M. A., Annu Rev Microbiol 56:371 (2002)).

West Nile virus (WNV) is a single-stranded positive sense RNA envelopevirus. It was first isolated and identified in the West Nile region ofUganda in 1937 from a febrile female adult (Smithburn, et al., Am J TropMed Hyg 3:9-18 (1954)). West Nile Virus has been classified as a memberof the family Flaviviridae using cross-neutralization tests withpolyclonal antisera (Boctor, et al., J. Virol Methods 26:305-311(1989)). West Nile virus is neuroinvasive (George, et al., Bull WHO62:879-882 (1984)); and severe human meningoencephalitis might occurconsequent to infection with West Nile virus, as seen in the outbreaksin North America (CDC, Update: West Nile Virus Encephalitis—New York1999, MMWR Morbid Mortal Wkly Rep 48:994-946; CDC, Update: West NileVirus Encephalitis—New York 1999. MMWR Morbid Mortal Wkly Rep51:1135-1136). During 1999-2002, WNV extended its range throughout muchof the eastern part of the United States, and its range within thewestern hemisphere is expected to continue to expand. Birds are thenatural reservoir hosts, and WNV is maintained in nature in amosquito-bird-mosquito transmission cycle primarily involving Culexspecies mosquitoes.

Recently, West Nile virus has emerged in temperate regions of Europe andNorth America, presenting a threat to public and animal health. The mostserious manifestation of WNV infection is fatal encephalitis(inflammation of the brain) in humans and horses, as well as mortalityin certain domestic and wild birds. West Nile virus infection has alsobeen a significant cause of human illness in the United States. Theenvelope glycoprotein of the West Nile virus (WNV-E) and otherflaviviruses may be important in formulating compositions to stimulatean immune response to generate neutralizing and protective antibodies.Currently, there are no compositions that prevent West Nile virusinfection, for example, by stimulating an immune response in a subject.

Japanese encephalitis (JE) virus is localized in Asia and northernAustralia (about 50,000 cases with about 10,000 deaths annually). Acomposition comprising an inactivated virus was recently associated witha case of acute disseminated encephalomyelitis, prompting the JapaneseMinistry of Health, Labor and Welfare to recommend the nationwidesuspension of compositions comprising inactivated virus.

The Dengue (DEN) disease is caused by four mosquito-borne, serologicallyrelated flaviviruses known as DEN-1 (also referred to herein as “Den1”or Den 1″), DEN-2 (also referred to herein as “Den2” or “Den 2”), DEN-3(also referred to herein as “Den3” or “Den 3”), and DEN-4 (also referredto herein as “Den4” or Den 4″), and is an important arboviral disease ofhumans. DEN is a major public health problem in all tropical areas ofthe world. About three billion people are at risk for DEN and about 50to about 100 million cases of dengue fever (DF) and hundreds ofthousands of cases of dengue hemorrhagic fever (DHF) occur in thetropics each year, including Mexico, the Caribbean and parts of Asia andthe South Pacific (Gubler, D. J., Ann Acad Med Singapore 27: 227-34(1998)). Dengue viruses are transmitted by peridomestic Aedes spp.mosquitoes, which inhabit the tropics, allowing endemicity of DF inthese areas. Infection by one virus causes Dengue Fever (DF), a febrileillness, which is not normally life-threatening, and leads to life-longprotective immunity against the infecting DEN serotype/virus. However,individuals that are infected by one serotype/virus remain susceptibleto infection by the other three DEN serotypes/viruses. Subsequentinfection by one of the other DEN serotypes/viruses can lead to Denguehemorrhagic fever (DHF) or dengue shock syndrome (DSS), which arelife-threatening diseases.

DHF may be the result of an antibody dependent enhancement (ADE) wherenon-neutralizing antibodies induced by the primary DEN infection formvirus-antibody complexes in secondary infections that are taken up intomacrophages by Fc receptors and, thus, enhance virus infection. About500,000 cases of DHF occur each year, mostly in children, with afatality rate of about 5%. About 600 million children are at risk forDEN infections, about 60 million may get DEN infections each year, andabout 60,000 may be hospitalized. In addition, to the public healthproblem, military personnel are often sent overseas to tropical areas ofthe world where DEN viruses are found. Significant numbers of soldierssuccumb to DEN while performing overseas, such as in Somalia, Grenada,Viet Nam and the Gulf conflicts. Attempts to develop a DEN vaccine haveproved difficult due to the need to develop a tetravalent vaccine thatprotects against all four DEN serotypes/viruses.

Methods to prevent flavivirus disease include vaccines to theflaviviruses (Barrett, A. D., Ann. N.Y. Acad. Sci. 951:262 (2001). Thesecompositions can be divided into two categories: live attenuated andinactivated. Compositions comprising live flavivirus have been developedfor YF and JE based on strains 17D and SA14-14-2, respectively, and werederived by empirical passage in chicken and hamster tissue,respectively. SA14-14-2 is produced in the People's Republic of China,grown in primary hamster kidney cell culture and very recently has beenapproved for use outside China. Both compositions are efficacious andrequire one or two doses, respectively, to develop protective immunity.There are inactivated virus compositions for JE and TBE. The inactivatedJE compositions are based on strains Nakayama, Beijing-1 or P3, whileinactivated TBE compositions are based on Central European TBE strainsNeudorfl and K23, and Russian Spring Summer encephalitis strains Sofjinand 205. These killed flavivirus compositions require about two doses(given about 1 week to about 2 months apart), a booster dose at aboutone year and periodic boosters about every 3 to about 4 years. Theantibody-mediated immunity, in particular neutralizing antibodies, maybe important in preventing flavivirus disease. Long-lived neutralizingantibody responses following administration of compositions to treatflavivirus disease or to prevent flavivirus disease may also beimportant.

Many different approaches have been taken to develop compositions toprevent flavivirus infection, but many have not been successful. Withrespect to the disease DEN, which is the result of four related viruses(DEN1, DEN2, DEN3, DEN4), a composition may need to be developed to oneor more the DEN flaviviruses. For example, a tetravalent (DEN1, DEN2,DEN3 and DEN4) composition may stimulate an immune responsesimultaneously against all four DEN viruses and thereby eliminate thepotential of antibody dependent enhancement.

Currently, there is no effective compositions to prevent infection bymany flaviviruses, including West Nile virus, Dengue virus, Tick-bornevirus, Kunjun virus, Murray Valley encephalitis virus and Yellow fevervirus (Chang, G. J., et al., Expert Rev. Vaccine 3:199 (2004)).Attenuation and immunogenicity may occur in compositions with liveattenuated flavivirus. Furthermore, compositions with tetravalent liveDengue flaviviruses may have problems with interference and imbalancedimmune response resulting in many compositions being tested withvariation in the quantity of each of the four DEN viruses. Compositionscomprising inactivated flavivirus may have problems with immunogenicityand the need for multiple doses. In addition, the production ofinactivated flavivirus compositions in infected mouse brains or cellculture can be complex, tedious, may result in unknown hazards if notproperly inactivated and may result in adverse effects when administeredto subjects. Thus, there is a need to develop new compositions for usein the prevention of flavivirus infection in subjects.

The compositions, fusion proteins and polypeptides of the inventionemploy pathogen-associated molecular patterns that trigger cellularevents resulting in the expression of costimulatory molecules, secretionof critical cytokines and chemokines; and efficient processing andpresentation of antigens to T-cells. As discussed above, TLRs recognizePAMPs including bacterial cell wall components (e.g., bacteriallipoproteins and lipopolysaccharides), bacterial DNA sequences thatcontain unmethylated CpG residues and bacterial flagellin. TLRs act asinitiators of the innate immune response and gatekeepers of the adaptiveimmune response (Medzhitov, R., et al., Cold Springs Harb. Symp. Quant.Biol. 64:429 (1999); Pasare, C., et al., Semin, Immunol 16:23 (2004);Medzhitov, R., et al., Nature 388:394 (1997); Barton, G. M., et al.,Curr. Opin. Immunol 14:380 (2002); Bendelac, A., et al., J. Exp. Med.195:F19 (2002)).

As discussed above, the binding of PAMPs to TLRs activates immunepathways for use in the compositions, fusion proteins and polypeptidesof the invention, which can be employed in stimulating the immune systemin a subject. The compositions, fusion proteins and polypeptides of theinvention can trigger an immune response to an antigen (e.g., a viralprotein, such as a flaviviral protein) and trigger signal transductionpathways of the innate and adaptive immune system of the subject tothereby stimulate the immune system of a subject. Stimulation of theimmune system of the subject may prevent infection by an antigen or avirus (e.g., a flavivirus) and thereby treat the subject or prevent thesubject from disease, illness and, possibly, death.

The compositions, fusion proteins and polypeptides of the invention, caninclude, for example, one, two, three, four, five, six or morepathogen-associated molecular patterns (e.g., Pam2Cys, Pam3Cys,flagellin) and one, two, three, four, five, six or more antigens. Whentwo or more PAMPs and/or two or more antigens and/or viral proteinscomprise the compositions, fusion proteins and polypeptides of theinvention, they are also referred to as “multimers.”

The pathogen-associated molecular pattern can be a TLR5 agonist (e.g.,at least a portion of at least one flagellin). The flagellin can be atleast one member selected from the group consisting of a fljB/STF2, anE. coli fliC, and a S. muenchen fliC. The flagellin can includefljB/STF2 (e.g., SEQ ID NO: 1) or a flagellin lacking a hinge region(e.g., SEQ ID NO: 3).

The pathogen-associated molecular pattern can be a TLR2 agonist. TheTLR2 agonist includes at least one member selected from the groupconsisting of a Pam2Cys and a Pam3Cys. Pam3Cys is([Palmitoyl]-Cys((RS)-2,3-di(palmitoyloxy)-propyl cysteine). Pam3Cys isalso referred to herein as “P2.” Pam2Cys is(S-[2,3-bis(palmitoyloxy)propyl]cysteine).

The viral protein for use in the compositions, fusion proteins andpolypeptides of the invention can be an envelope protein of at least onemember selected from the group consisting of a West Nile viral envelopeprotein, a Langat viral envelope protein, a Kunjin viral envelopeprotein, a Murray Valley encephalitis viral envelope protein, a Japaneseencephalitis viral envelope protein, a Tick-borne encephalitis viralenvelope protein, a Yellow fever viral envelope protein and a Dengueviral envelope protein.

The compositions, fusion proteins and polypeptides of the invention canemploy any portion of the envelope protein of a flavivirus. Thecompositions, fusion proteins and polypeptides of the invention caninclude at least a portion of at least one member selected from thegroup consisting of domain I, domain II and domain III of an envelopeprotein of a flavivirus. “At least a portion,” as used herein, inreference to a domain of an envelope protein, means any part of theenvelope protein domain or the entirety of the envelope protein. Forexample, SEQ ID NOS: 88 and 100-151, include at least a portion ofdomain III of the West Nile virus envelope protein.

“EI,” “EII,” and “EIII,” as used herein, refer to domains I, II and III,respectively, of the West Nile flavivirus envelope protein. “JEI,”“JEII,” and “JEIII,” as used herein, refer to domains I, II and III,respectively, of the Japanese encephalitis flavivirus envelope protein.“Den1 I,” “Den1 II,” and “Den1 III,” as used herein refer to domains I,II and III, respectively, of the Dengue 1 flavivirus envelope protein.Likewise, designations for the domains of envelope proteins of otherflaviviruses are referenced by the flavivirus name followed by thedomain number (e.g., (Tick-borne) TBI (Tick-borne), TBII, TBIII, Den2 I,Den2 II, Den2 III).

The portion of an envelope protein of a flavivirus employed in thecompositions, fusion proteins and polypeptides of the invention caninclude at least one member selected from the group consisting of atleast a portion of domain I, at least a portion of domain II and atleast a portion of domain III. When a domain is designated with a “+,”for example “EIII+” or “JEIII+,” the portion of the envelope proteinreferenced as “III” is one component of the total of that domain plus atleast one of at least a portion of either or both of domains I and II.For example, “EIII+,” as used herein, means the compositions, fusionproteins and polypeptides of the invention include domain III and atleast a portion of domain I. “EIII+” is also referred to as “EI/III.”“JEIII+” is also referred to as “JEI/III.” Similarly, when compositions,fusion proteins and polypeptides of the invention include domains ofenvelope proteins of flavivirus, the domains can be any combination ofdomains I, II, and III and can be designated based on the domain. Forexample, EI/II includes domain I and II of the West Nile flavivirus. Theabsence of a “+” in reference to a domain (e.g., EIII, JEIII, Den1 III)of an envelope protein employed in the compositions, fusion proteins andpolypeptides of the invention means that the composition, fusion proteinand polypeptide includes the referenced domain. For example, “Den1 III”means the compositions, fusion proteins and compositions include domainIII, not domains I and II, of the Dengue 1 virus.

The West Nile viral envelope protein for use in the compositions, fusionproteins and polypeptides of the invention can include at least aportion of at least one member selected from the group consisting ofMEKLQLKGTTYGVCSKAFKFLGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIGK (SEQ ID NO: 7,which is an EIII+ amino acid sequence, the italicized amino acids aredomain I of the envelope protein and the remaining sequence is domainIII of the envelope protein);GTTYGVCSKAFKFARTPADTGHGTVVLELQYTGKDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANSKVLIELEPPFGDSYIVVGRGEQQINHHWHKSG (SEQ ID NO: 8, West Nilevirus, Stanford, Conn., also referred to as “West Nile S”);GTTYGVCSKAFKFLGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIELEPPFGDSYVVGRGEQQINHHWHKSG (SEQ ID NO: 9, West Nilevirus, New York, N.Y., also referred to as “West Nile NY”); andELEPPFGDSYIVVGRGEQQINHHWHKS (SEQ ID NO: 10). SEQ ID NO: 7 is encoded byATGGAAAAATTGCAGTTGAAGGGAACAACCTATGGCGTCTGTTCAAAGGCTTTCAAGTTTCTTGGGACTCCCGCAGACACAGGTCACGGCACTGTGGTGTTGGAATTGCAGTACACTGGCACGGATGGACCTTGCAAAGTTCCTATCTCGTCAGTGGCTTCATTGAACGACCTAACGCCAGTGGGCAGATTGGTCACTGTCAACCCTTTTGTTTCAGTGGCCACGGCCAACGCTAAGGTCCTGATTGAATTGGAACCACCCTTTGGAGACTCATACATAGTGGTGGGCAGAGGAGAACAACAGATCAATCACCATTGGCACAAGTCTGGAAGCAGCATTG GCAAA (SEQ IDNO: 11). The West Nile viral envelope protein can include a protein thathas at least about 70% identity, at least about 75% identity, at leastabout 80% identity, at least about 85% identity, at least about 90%identity, at least about 95% identity and at least about 99% identity toa polypeptide that includes SEQ ID NO: 7, which include portions ofdomains I and III (referred to herein as “EIII+”) of the West Nilevirus.

The Langat virus envelope protein for use in the compositions, fusionproteins and polypeptides of the invention can include at least aportion of GLTYTVCDKTKFTWKRAPTD SGHDTVVMEVGFSGTRPCRIPVRAVAHGVPEVNVAMLITPNPTMENNGGGFIEMQLPPGDNIIYVGDLNHQWFQKG (SEQ ID NO: 12). The Kunjin virusenvelope protein can include at least a portion ofGTTYGVCSKAFRFLGTPADTGHGTVVLELQYTGTDGPCKIPISSVASLNDLTPVGRLVTVNPFVSVSTANAKVLIELEPPFGDSYIVVGRGEQQINHHWHKSG (SEQ ID NO: 13). The MurrayValley encephalitis envelope protein can include at least a portion ofGTTYGMCTEKFTFSKNPADTGHGTVVLELQYTGSDGPCKIPISSVASLNDMTPVGRMVTANPYVASSTANAKVLVEIEPPFGDSYIVVGRGDKQINHHWHKEG (SEQ ID NO: 14). TheJapanese encephalitis envelope protein can include at least one memberselected from the group consisting of at least a portion ofGTTYGMCTEKFSFAKNPADTGHGTVVIELSYSGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGSDYIVVGMGDKQINHHWHKAG (SEQ ID NO: 15) andEMEPPFGDSYIVVMGDKQINHHWHKA (SEQ ID NO: 16). The Tick-borne encephalitisenvelope protein can include at least a portion ofGLTYTMCDKTKFTWKRAPTDSGHDTVVMEVTFSGTKPCRIPVRAVAHGSPDVNVAMLITPNPTIENNGGGFIEMQLPPGDNIIYVGELSHQWFQK (SEQ ID NO: 17). The Yellow fevervirus envelope protein can include at least a portion ofGLTYTMCDKTFTWKRAPTDSGHDTVVMEVTFSGTKPCRIPVRAVAHGSPDVNVAMLITPNPTIENNGGGFIEMQLPPGDNIIYVGELSHQWFQK (SEQ ID NO: 18). The envelopeprotein of a flavivirus can include at least a portion of at least onemember selected from the group consisting ofGTTYGMCSKKFTFRPADTGHGTVVLELQYSGDGPCKIPISVASKNDLTPVGRLVTVNPFVSSTANAKVLIEMEPPFGDSYIVVGGEQINHHWHKG (SEQ ID NO: 19) andGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKK (SEQ ID NO: 40). SEQ ID NOS: 12,13, 14, 15, 16, 17, 18, 19 and 40 are portions of domain III of theviral envelope protein.

In another embodiment, the invention is a composition comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one Den2 viral envelope protein,wherein the Den2 viral envelope protein is at least one member selectedfrom the group consisting of EAEPPFGDSYIIIGVEPQQLKLNWFKK (SEQ ID NO:22), SEQ ID NO: 40 and SEQ ID NO: 97.

The compositions, fusion proteins and polypeptides of the invention caninclude Den 1 (EAEPPFGESYIVVGAGEKALKLSWFKK (SEQ ID NO.: 20); Den 1 PR 94(Puerto Rico, 1994) (ETEPPFGESYIVVGAGEKALKLSWFKK (SEQ ID NO: 21)); Den 3(EAEPPFGESNIVIGIGDKALKINWYKK (SEQ ID NO: 23)); and Den 4(ELEPPFGESYIVIGVGNSALTLHWFRK (SEQ ID NO: 24)). SEQ ID NOS: 20, 21, 22,23 and 24 are portions of domain III of Den1, Den2, Den3 and Den4flaviviruses. At least a portion of domain III of the four Dengueviruses can be employed together or separately in the compositions,fusion proteins or polypeptides of the invention. For example, domainIII of Den1 (strain 16007), Den2 (strain 516803), Den3 (strain H53489)and Den4 (strain 703) can be employed separately or in combination. Thepathogen-associated molecular pattern and Den2 envelope viral proteincan be components of a fusion protein.

In still another embodiment, the invention is a composition comprisingat least a portion of at least one pathogen-associated molecular patternand at least a portion of at least one member selected from the groupconsisting of a Den1 viral envelope protein, a Den2 viral envelopeprotein, a Den3 viral envelope protein and a Den4 viral envelopeprotein.

In an additional embodiment, the invention is a fusion proteincomprising at least a portion of at least one pathogen-associatedmolecular pattern and at least a portion of at least one viral proteinselected from the group consisting of a West Nile viral protein, aLangat viral protein, a Kunjin viral protein, a Murray Valleyencephalitis viral protein, a Japanese encephalitis viral protein, aTick-borne encephalitis viral protein, a Yellow fever viral protein anda hepatitis C viral protein. The hepatitis C viral protein for use inthe compositions, fusion proteins and polypeptides of the invention caninclude a polypeptide of at least one member selected from the groupconsisting of SEQ ID NO: 64 (FIG. 22) and SEQ ID NO: 65 (FIG. 23), whichare encoded by SEQ ID NOS: 66 (FIGS. 24) and 67 (FIG. 25), respectively.

In another embodiment, the invention is a fusion protein comprising atleast a portion of at least one pathogen-associated molecular patternand at least a portion of at least one member selected from the groupconsisting of a Den1 viral envelope protein, a Den2 viral envelopeprotein, a Den3 viral envelope protein and a Den4 viral envelopeprotein.

Fusion proteins of the invention can further include a linker betweenthe pathogen-associated molecular pattern and the viral protein. Thelinker can be an amino acid linker. The amino acid linker can includesynthetic or naturally occurring amino acid residues. The amino acidlinker employed in the fusion proteins of the invention can include atleast one member selected from the group consisting of a lysine residue,a glutamic acid residue, a serine residue, a cysteine residue and anarginine residue. “Amino acid linker,” as used herein, is also referredto as a “peptide linker” The amino acid linker can include at least onemember selected from the group consisting of a peptide ofKGNSKLEGQLEFPRTS (SEQ ID NO: 26); EFCRYPAQWRPL (SEQ ID NO: 28);EFSRYPAQWRPL (SEQ ID NO: 60);KGNSKLEGQLEFPRTSPVWWNSADIQHSGGRQCDGYLQNSPLRPL (SEQ ID NO: 62);EFSRYPAQWRPL (SEQ ID NO: 75); which are encoded byAAGGGCAATTCGAAGCTTGAAGGTCAATTGGAATTCCCTAGGACTAGT (SEQ ID NO: 25);GAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTC (SEQ ID NO: 27);GAATTCTCTAGATATCCAGCACAGTGGCGGCCGCTC (SEQ ID NO: 61);AAGGGCAATTCGAAGCTTGAAGGTCAATTGGAATTCCCTAGGACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGCGGCCGCTC (SEQ ID NO: 63); andGAATTCTCTAGATATCCAGCACAGTGGCGGCCGCT ((SEQ ID NO: 74).

The fusion proteins of the invention can further include a linkerbetween at least one component of the fusion protein (e.g., Pam3Cys,Pam2Cys, flagellin, PAMP) and at least one other component of the fusionprotein (e.g., at least a portion of an antigen, at least a portion of aviral protein) of the composition, a linker between at least two ofsimilar components of the fusion protein (e.g., Pam3Cys, Pam2Cys,flagellin, PAMP, at least a portion of an antigen, at least a portion ofa viral protein) or any combination thereof.

“Linker,” as used herein in reference to a fusion protein of theinvention, refers to a connector between components of the fusionprotein in a manner that the components of the fusion protein are notdirectly joined. For example, one component of the fusion protein (e.g.,Pam3Cys, Pam2Cys, PAMP) can be linked to a distinct component (e.g., atleast a portion of an antigen, at least a portion of a viral protein) ofthe fusion protein. Likewise, at least two or more similar or likecomponents of the fusion protein can be linked (e.g., two PAMPs canfurther include a linker between each PAMP, or two antigens can furtherinclude a linker between each antigen, or two viral proteins can furtherinclude a linker between each viral protein).

Additionally or alternatively, the fusion proteins of the invention caninclude a combination of a linker between distinct components of thefusion protein and similar or like components of the fusion protein. Forexample, a fusion protein can comprise at least two PAMPs, Pam3Cysand/or Pam2Cys components that further includes a linker between, forexample, two or more PAMPs; at least two antigens or at least two viralproteins that further include a linker between them; a linker betweenone component of the fusion protein (e.g., PAMP) and another distinctcomponent of the fusion protein (e.g., at least a portion of an antigen,at least a portion of a viral protein), or any combination thereof.Thus, the fusion proteins of the invention can further include a linkerbetween at least two pathogen-associated molecular patterns, a linkerbetween at least two antigens, a linker between at least two viralproteins, or any combination thereof.

The pathogen-associated molecular pattern of the fusion proteins of theinvention can be fused to a carboxy-terminus, an amino-terminus or botha carboxy- and an amino-terminus of an antigen or at least a portion ofa viral protein (e.g., a flavivirus viral protein, such as at least aportion of domain III of the West Nile envelope protein, referred to as“EIII,” at least a portion of domain III of Dengue1 envelope protein)referred to as “Den1 III.” “Fused to,” as used herein, means covalentlyor noncovalently linked or recombinantly produced together.

The fusion proteins of the invention can include at least onepathogen-associated molecular pattern between at least two antigens orat least two viral proteins, which can, optionally, include a linkerbetween the pathogen-associate molecular pattern and the antigen or theviral protein. The fusion proteins of the invention can include apathogen-associated molecular pattern fused between at least two viralproteins (e.g., designated as “viral protein.PAMP.viral protein”). Thefusion proteins of the invention can include an antigen or a viralprotein fused between at least two pathogen-associated molecularpatterns (e.g., designated as “PAMP.viral protein.PAMP”).

The pathogen-associated molecular pattern of the fusion proteins of theinvention can be a TLR5 agonist, such as a flagellin. The antigen orviral protein of the fusion proteins of the invention can be fused tothe flagellin in a region of the flagellin that lacks at least a portionof a hinge region of the flagellin. For example, at least a portion ofthe hinge region of the fljB/STF2 flagellin of SEQ ID NO: 1 (FIG. 1) canbe deleted and an antigen or a viral protein can be fused to theflagellin in the region of the deletion.

The antigen or viral protein of the fusion proteins of the invention canbe fused to the flagellin in a region of the flagellin that contains ahinge region of the flagellin. For example, an antigen or viral proteincan be fused to the fljB/STF2 flagellin of SEQ ID NO: 1 (FIG. 1) at anyplace in the hinge region, for example, at any place with amino acids176-415 of SEQ ID NO: 1.

The antigen or viral protein of the fusion proteins of the invention canbe fused to the flagellin in a region of the flagellin that lacks ahinge region of the flagellin, wherein the hinge region has beenreplaced with an artificial hinge region, such as an amino acid linker.For example, an antigen or viral protein can be fused to the fljB/STF2Δflagellin of SEQ ID NO: 3 (FIG. 3) by placing an amino acid linker (alsoreferred to herein as an “artificial hinge” or “an artificial hingeregion” or “an artificial hypervariable region”), as depicted, forexample, with the placement of an amino acid linker (HGAPVDPASPW, SEQ IDNO: 183) between amino acids 175 to 186 of SEQ ID NO: 3.

In another embodiment, the invention is a fusion protein comprising atleast a portion of at least one member selected from the groupconsisting of fljB/STF2, an E. coli fliC, and a S. muenchen fliC and atleast a portion of at least one member selected from the groupconsisting of a Den1 viral envelope protein, a Den2 viral envelopeprotein, a Den3 viral envelope protein and a Den4 viral envelopeprotein. The portion of the envelope protein can be at least a portionof at least one member selected from the group consisting of domain I,domain II and domain III of the envelope protein.

In still another embodiment, the invention includes a polypeptide thatincludes SEQ ID NOS: 71, 72, 30, 32, 34, 36, 38, 55, 76, 6, 80, 82, 84,86 and 159 and a polypeptide encoded by SEQ ID NOS: 70, 73, 29, 31, 33,35, 37, 54, 77, 5, 81, 83, 85, 87 and 158.

In an additional embodiment, the invention includes a polypeptide havingat least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%and at least about 99% sequence identity to the polypeptides of SEQ IDNOS: 71, 72, 30, 32, 34, 36, 38, 55, 75, 6, 80, 82, 84, 86 and 159 andthe nucleic acids of SEQ ID NOS: 70, 73, 29, 31, 33, 35, 37, 54, 77, 5,81, 83, 85, 87 and 158.

In a further embodiment, the invention includes compositions, fusionproteins and polypeptides that include a polypeptide having a flagellinthat includes polypeptides having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98% and at least about 99% sequenceidentity to the polypeptides of SEQ ID NOS: 1, 3, 58 and 68 and thenucleic acids of 2, 4, 59 and 69.

The percent identity of two amino acid sequences (or two nucleic acidsequences) can be determined by aligning the sequences for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst sequence). The amino acid sequence or nucleic acid sequences atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). The length of the protein ornucleic acid encoding a PAMP, at least a portion of a fusion protein, aviral protein, or a polypeptide of the invention aligned for comparisonpurposes is at least 30%, preferably, at least 40%, more preferably, atleast 60%, and even more preferably, at least 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% or 100%, of the length of the reference sequence, forexample, the nucleic acid sequence of a PAMP, at least a portion of aviral protein, or a polypeptide or fusion protein, for example, asdepicted in SEQ ID NOS: 71, 72, 30, 32, 34, 36, 38, 55, 75, 6, 80, 82,84, 86 and 159.

The actual comparison of the two sequences can be accomplished bywell-known methods, for example, using a mathematical algorithm. Apreferred, non-limiting example of such a mathematical algorithm isdescribed in Karlin et al. (Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993), the teachings of which are hereby incorporated by reference inits entirety). Such an algorithm is incorporated into the BLASTN andBLASTX programs (version 2.2) as described in Schaffer et al. (NucleicAcids Res., 29:2994-3005 (2001), the teachings of which are herebyincorporated by reference in its entirety). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN; available at the Internet site for the National Centerfor Biotechnology Information) can be used. In one embodiment, thedatabase searched is a non-redundant (NR) database, and parameters forsequence comparison can be set at: no filters; Expect value of 10; WordSize of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11and an Extension of 1.

Another mathematical algorithm employed for the comparison of sequencesis the algorithm of Myers and Miller, CABIOS (1989), the teachings ofwhich are hereby incorporated by reference in its entirety. Such analgorithm is incorporated into the ALIGN program (version 2.0), which ispart of the GCG (Accelrys, San Diego, Calif.) sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 is used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti (Comput. Appl. Biosci., 10: 3-5 (1994), theteachings of which are hereby incorporated by reference in itsentirety); and FASTA described in Pearson and Lipman (Proc. Natl. Acad.Sci. USA, 85: 2444-2448 (1988), the teachings of which are herebyincorporated by reference in its entirety).

The percent identity between two amino acid sequences can also beaccomplished using the GAP program in the GCG software package(Accelrys, San Diego, Calif.) using either a Blossom 63 matrix or aPAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. In yet another embodiment, the percent identitybetween two nucleic acid sequences can be accomplished using the GAPprogram in the GCG software package (Accelrys, San Diego, Calif.), usinga gap weight of 50 and a length weight of 3.

The nucleic acid sequence encoding a PAMP, at least a portion of a viralprotein, fusion proteins of the invention and polypeptides of theinvention can include nucleic acid sequences that hybridize to, forexample, a fljB/STF2 (e.g., SEQ ID NOS: 2, 4), a fliC (e.g., SEQ ID NOs:59, 69), at least a portion of a viral protein (e.g., SEQ ID NOS: 39,160, 162, 164, 166 and 177 and fusion proteins of the invention (e.g.,SEQ ID NOS: 71, 72, 30, 32, 34, 36, 38, 55, 75, 6, 80, 82, 84 and 86)under selective hybridization conditions (e.g., highly stringenthybridization conditions). As used herein, the terms “hybridizes underlow stringency,” “hybridizes under medium stringency,” “hybridizes underhigh stringency,” or “hybridizes under very high stringency conditions,”describe conditions for hybridization and washing of the nucleic acidsequences. Guidance for performing hybridization reactions, which caninclude aqueous and nonaqueous methods, can be found in Aubusel, F. M.,et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(2001), the teachings of which are hereby incorporated herein in itsentirety.

For applications that require high selectivity, relatively highstringency conditions to form hybrids can be employed. In solutions usedfor some membrane based hybridizations, addition of an organic solvent,such as formamide, allows the reaction to occur at a lower temperature.High stringency conditions are, for example, relatively low salt and/orhigh temperature conditions. High stringency are provided by about 0.02M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C.High stringency conditions allow for limited numbers of mismatchesbetween the two sequences. In order to achieve less stringentconditions, the salt concentration may be increased and/or thetemperature may be decreased. Medium stringency conditions are achievedat a salt concentration of about 0.1 to 0.25 M NaCl and a temperature ofabout 37° C. to about 55° C., while low stringency conditions areachieved at a salt concentration of about 0.15 M to about 0.9 M NaCl,and a temperature ranging from about 20° C. to about 55° C. Selection ofcomponents and conditions for hybridization are well known to thoseskilled in the art and are reviewed in Ausubel et al. (1997, ShortProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units2.8-2.11, 3.18-3.19 and 4-64.9).

In yet another embodiment, the invention is a composition comprising atleast one Pam3Cys and at least a portion of at least one flavivirusprotein (e.g., at least one member selected from the group consisting ofa West Nile viral protein, a Dengue viral protein, a Langat viralprotein, a Kunjin viral protein, a Murray Valley encephalitis viralprotein, a Japanese encephalitis viral protein, a Tick-borneencephalitis viral protein, a Yellow fever viral protein and a hepatitisC viral protein). The Dengue viral protein can be at least one memberselected from the group consisting of a Den1 viral protein, a Den2 viralprotein, a Den3 viral protein and a Den4 viral protein. The Pam3Cys andthe flavivirus protein can be components of a fusion protein.

An additional embodiment of the invention is a composition comprising atleast one Pam2Cys and at least a portion of at least one flavivirusprotein (e.g., at least one member selected from the group consisting ofa West Nile viral protein, a Dengue viral protein, a Langat viralprotein, a Kunjin viral protein, a Murray Valley encephalitis viralprotein, a Japanese encephalitis viral protein, a Tick-borneencephalitis viral protein, a Yellow fever viral protein and a hepatitisC viral protein). The Dengue viral protein can be at least one memberselected from the group consisting of a Den1 viral protein, a Den2 viralprotein, a Den3 viral protein and a Den4 viral protein. The Pam2Cys andthe flavivirus protein can be components of a fusion protein.

In still another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes the compositions, fusionproteins and polypeptides of the invention.

“Stimulating an immune response,” as used herein, refers to thegeneration of antibodies to at least a portion of an antigen or a viralprotein (e.g., a West Nile viral protein, a Dengue viral protein, aLangat viral protein, a Kunjin viral protein, a Murray Valleyencephalitis viral protein, a Japanese encephalitis viral protein, aTick-borne encephalitis viral protein, a Yellow fever viral protein anda hepatitis C viral protein). Stimulating an immune response in asubject can include the production of humoral and/or cellular immuneresponses that are reactive against the antigen or viral protein. Instimulating an immune response in the subject, the subject may beprotected from infection by the antigen or virus or conditionsassociated with infection by the antigen or virus that may diminish orbe halted as a consequence of stimulating an immune response in thesubject.

The compositions, fusion proteins and polypeptides of the invention foruse in methods to stimulate immune responses in subjects, can beevaluated for the ability to stimulate an immune response in a subjectusing well-established methods. Exemplary methods to determine whetherthe compositions, fusion proteins and polypeptides of the inventionstimulate an immune response in a subject, include measuring theproduction of antibodies specific to the antigen or viral protein (e.g.,IgG antibodies) by a suitable technique such as, ELISA assays; thepotential to induce antibody-dependent enhancement (ADE) of a secondaryinfection; macrophage-like assays; neutralization assessed by using thePlaque Reduction Neutralization Test (PRNT₈₀); the ability to generateserum antibodies in non-human models (e.g., mice, rabbits, monkeys)(Putnak, et al., Vaccine 23:4442-4452 (2005)); the ability to survive achallenge of exposure to an antigen, in particular, a viral antigenemploying non-human animals, such as mice and monkeys.

A “subject,” as used herein, can be a mammal, such as a primate orrodent (e.g., rat, mouse). In a particular embodiment, the subject is ahuman.

In a further embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one viral protein selected from the group consisting of a WestNile viral protein, a Langat viral protein, a Kunjin viral protein, aMurray Valley encephalitis viral protein, a Japanese encephalitis viralprotein, a Tick-borne encephalitis viral protein, and a Yellow fevervirus protein.

In yet another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one Den2 envelope protein, wherein the Den2 envelope protein isselected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 40 andSEQ ID NO: 97.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a Tick-borne encephalitis viral protein and a Yellowfever viral protein.

In still another embodiment, the invention is a method stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes at least a portion of at leastone pathogen-associated molecular pattern and at least a portion of atleast one member selected from the group consisting of a Den1 viralenvelope protein, a Den2 viral envelope protein, a Den3 viral envelopeprotein and a Den4 viral envelope protein.

An additional embodiment of the invention is a method stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one member selected from the group consisting of a fljB/STF2, anE. coli fliC, and a S. muenchen fliC and at least a portion of at leastone member selected from the group consisting of a Den1 viral envelopeprotein (e.g.,KGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGENYIVVGAGEKALKLSWFKK (SEQ ID NOS: 21 and 96)), aDen2 viral envelope protein (e.g., SEQ ID NOS: 22, 40 andKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKTPFEIMDLEKRHVLGRLTTVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLDWFKK (SEQ ID NO: 97)), a Den3 viralenvelope protein (e.g., SEQ ID NO: 23 andKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYRK (SEQ ID NO: 98)) and a Den4viral envelope protein (e.g., SEQ ID NO: 24 andKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISPTPFAENTNSVTNIELERPLDSYIVIGVGDSALTLHWFRK (SEQ ID NO: 99)).

In a further embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In yet another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition comprising at least a portion of at least oneantigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lack at least a portion of a hinge region.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein comprising at least a portion of at leastone antigen and at least a portion of at least one flagellin, wherein atleast one of the flaggelins lack at least a portion of a hinge region.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a fusion protein comprising at least a portion of at leastone antigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lack at least a portion of the hinge region.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a Tick-borne encephalitis viral protein, and a Yellowfever virus protein.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one Den2 envelope protein, wherein the Den2 envelope proteinis at least one member selected from the group consisting of SEQ ID NO:22, SEQ ID NO: 40 and SEQ ID NO: 97.

In still another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one viral protein selected from the group consisting of aWest Nile viral protein, a Langat viral protein, a Kunjin viral protein,a Murray Valley encephalitis viral protein, a Japanese encephalitisviral protein, a Tick-borne encephalitis viral protein and a Yellowfever viral protein.

In an additional embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one member selected from the group consisting of a Salmonellatyphimurium flagellin type 2 (fljB/STF2), an E. coli fliC, and a S.muenchen fliC and at least a portion of at least one member selectedfrom the group consisting of a Den1 viral envelope protein, a Den2 viralenvelope protein, a Den3 viral envelope protein and a Den4 viralenvelope protein.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein that includes at least a portion of atleast one pathogen-associated molecular pattern and at least a portionof at least one member selected from the group consisting of a Den1viral envelope protein, a Den2 viral envelope protein, a Den3 viralenvelope protein and a Den4 viral envelope protein.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition comprising at least a portion of at leastone antigen and at least a portion of at least one flagellin, wherein atleast one of the flagellins lacks at least a portion of a hinge region.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a fusion protein comprising at least a portion of atleast one antigen and at least a portion of at least one flagellin,wherein at least one of the flagellins lacks at least a portion of ahinge region.

“Stimulates a protective immune response,” as used herein, meansadministration of the compositions of the invention, such as the fusionproteins (e.g., fusion proteins that include a TLR agonist and at leasta portion of a flavivirus), results in production of antibodies to theprotein to thereby cause a subject to survive challenge by an otherwiselethal dose of a viral protein, such as a flavivirus. Techniques todetermine a lethal dose of a virus (e.g., a flavivirus) are known to oneof skill in the art (see, for example, Harmon, M. W., et al., J. Clin.Microbiol. 26:333-337 (1988); Reed, L. J., et al., Am. J. Hyg.27:493-497 (1938); Rose, T., et al., J. Clin. Microbiol. 37:937-943(1999); Walls, H. H. et al., J. Clin. Microbiol. 23:240-245 (1986);Current Protocols in Immunology, 19.11.1-19.11.32, Cottey, R., et al.,John Wiley & Sons, Inc (2001)). Exemplary techniques for determining alethal dose can include administration of varying doses of virus and adetermination of the percent of subjects that survive followingadministration of the dose of virus (e.g., LD₁₀, LD₂₀, LD₄₀, LD₅₀, LD₆₀,LD₇₀, LD₈₀, LD₉₀). For example, a lethal dose of a virus that results inthe death of 50% of a population of subjects is referred to as an“LD₅₀”; a lethal dose of a virus that results in the death of 80% of apopulation of subjects is referred to herein as “LD₈₀”; a lethal dose ofa virus that results in death of 90% of a population of subjects isreferred to herein as “LD₉₀.”

For example, determination of the LD₉₀ can be conducted in subjects(e.g., mice) by administering intranasally or intrapentoneally varyingdoses (e.g., dilutions, such as log and half-log dilutions of plagueforming units (pfu) (e.g., 10 pfu) followed by an assessment of thesurvival of the subjects about 14 days to about 21 days after infectionwith the virus. Protective immunity can be assessed by physicalappearance of the subject, general demeanor (active), weight (initialloss of weight followed by return to a weight about the weight of thesubject prior to infection with the virus) and survival after about 14to about 21 days following infection with the flavivirus.

Assessment of stimulation of protective immunity can also be made byemploying assays that assess the ability of the antibodies produced inresponse to the compositions of the invention (e.g., a portion of aflavivirus, such as a protein portion of West Nile virus, JE virus orDengue virus) to result in survival of the subject (see, for example,Current Protocols in Immunonology, 19.11.1-19.11.32, Cottey, R., et al.,John Wiley & Sons, Inc (2001)).

In another embodiment, the invention is a method of making fusionproteins or components of fusion proteins (e.g., TLR agonists, at leasta portion of a flavivirus) described herein. Methods for making fusionproteins and the components of fusion proteins can include production offusion proteins in host cells (e.g., eukaroytic host cells, prokaryotichost cells) by, for example, transfecting or transforming host cellswith nucleic acid constructs encoding the fusion proteins or componentsof the fusion proteins.

The methods of making a protein that stimulates an immune response orstimulates a protective immune response in a subject can further includethe step of deleting at least one glycosylation site in the nucleic acidsequence encoding the PAMP, TRL agonist or antigen (e.g., flavivirus).The glycosylation site that is deleted can include an N-glycosylationsite or an O-glycosylation site.

The host cell employed in the methods described herein can be aprokaryotic host cell. The prokaryotic host cell can be at least onemember selected from the group consisting of an E. coli prokaryotic hostcell, a Pseudomonas prokaryotic host cell, a Bacillus prokaryotic hostcell, a Salmonella prokaryotic host cell and a P. fluorescensprokaryotic host cell.

The eukaryotic host cells employed in the methods of the invention caninclude a Saccharomyces eukaryotic host cell, an insect eukaryotic hostcell (e.g., at least one member selected from the group consisting of aBaculovirus infected insect cell, such as Spodoptera frugiperda (Sf9) orTrichhoplusia ni (High5) cells; and a Drosophila insect cell, such asDme12 cells), a fungal eukaryotic host cell, a parasite eukaryotic hostcell (e.g., a Leishmania tarentolae eukaryotic host cell), CHO cells,yeast cells (e.g., Pichia) and a Kluyveromyces lactis host cell.

Suitable eukaryotic host cells and vectors can also include plant cells(e.g., tomato; chloroplast; mono- and dicotyledonous plant cells;Arabidopsis thaliana; Hordeum vulgare; Zea mays; potato, such as Solanumtuberosum; carrot, such as Daucus carota L.; and tobacco, such asNicotiana tabacum, Nicotiana benthamiana (Gils, M., et al., PlantBiotechnol J. 3:613-20 (2005); He, D. M., et al., Colloids Surf BBiointerfaces, (2006); Huang, Z., et al., Vaccine 19:2163-71 (2001);Khandelwal, A., et al., Virology. 308:207-15 (2003); Marquet-Blouin, E.,et al., Plant Mol Biol 51:459-69 (2003); Sudarshana, M. R., et al. PlantBiotechnol J. 4:551-9 (2006); Varsani, A., et al., Virus Res, 120:91-6(2006); Kamarajugadda S., et al., Expert Rev Vaccines 5:839-49 (2006);Koya V, et al., Infect Immun. 73:8266-74 (2005); Zhang, X., et al.,Plant Biotechnol J. 4:419-32 (2006)).

The proteins made by the methods of the invention and the compositionsof the invention can be purified and characterized employing well-knownmethods (e.g., gel chromatography, cation exchange chromatography,SDS-PAGE), as described herein.

In a further embodiment, the invention is the host cells and vectorsthat include the nucleic acid sequences of the invention or encodedfusion proteins of the invention.

An “effective amount,” when referring to the amount of a composition,fusion protein or a polypeptide of the invention, refers to that amountor dose of the composition, fusion protein, or a polypeptide, that, whenadministered to the subject is an amount sufficient for therapeuticefficacy (e.g., an amount sufficient to stimulate an immune response inthe subject). The compositions, fusion proteins, or polypeptides of theinvention can be administered in a single dose or in multiple doses.

The methods of the present invention can be accomplished by theadministration of the compositions, fusion proteins or polypeptides ofthe invention by enteral or parenteral means. Specifically, the route ofadministration is by oral ingestion (e.g., drink, tablet, capsule form)or intramuscular injection of the composition, fusion protein orpolypeptide. Other routes of administration as also encompassed by thepresent invention including intravenous, intradermal, intraarterial,intraperitoneal, or subcutaneous routes, and nasal administration.Suppositories or transdermal patches can also be employed.

The compositions, fusion proteins or polypeptides of the invention canbe administered ex vivo to a subject's autologous dendritic cells.Following exposure of the dendritic cells to the composition, fusionprotein or polypeptide of the invention, the dendritic cells can beadministered to the subject.

The compositions, fusion proteins or polypeptides of the invention canbe administered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the composition, fusion protein or polypeptide of theinvention individually or in combination. Where the composition, fusionprotein or polypeptide are administered individually, the mode ofadministration can be conducted sufficiently close in time to each other(for example, administration of the composition close in time toadministration of the fusion protein) so that the effects on stimulatingan immune response in a subject are maximal. It is also envisioned thatmultiple routes of administration (e.g., intramuscular, oral,transdermal) can be used to administer the compositions and fusionproteins of the invention.

The compositions, fusion proteins or polypeptide of the invention can beadministered alone or as admixtures with conventional excipients, forexample, pharmaceutically, or physiologically, acceptable organic, orinorganic carrier substances suitable for enteral or parenteralapplication which do not deleteriously react with the extract. Suitablepharmaceutically acceptable carriers include water, salt solutions (suchas Ringer's solution), alcohols, oils, gelatins and carbohydrates suchas lactose, amylose or starch, fatty acid esters, hydroxymethycellulose,and polyvinyl pyrrolidine. Such preparations can be sterilized and, ifdesired, mixed with auxillary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the compositions, fusion proteinsor polypeptides of the invention. The preparations can also be combined,when desired, with other active substances to reduce metabolicdegradation. The compositions, fusion proteins or polypeptides of theinvention can be administered by is oral administration, such as adrink, intramuscular or intraperitoneal injection. The compositions,fusion proteins, or polypeptides alone, or when combined with anadmixture, can be administered in a single or in more than one dose overa period of time to confer the desired effect (e.g., alleviate preventflavivirus infection, to alleviate symptoms of flavivirus infection).

When parenteral application is needed or desired, particularly suitableadmixtures for the compositions, fusion proteins or polypeptides areinjectable, sterile solutions, preferably oily or aqueous solutions, aswell as suspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and thelike. Ampules are convenient unit dosages. The compositions, fusionproteins or polypeptides can also be incorporated into liposomes oradministered via transdermal pumps or patches. Pharmaceutical admixturessuitable for use in the present invention are well-known to those ofskill in the art and are described, for example, in PharmaceuticalSciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309 theteachings of which are hereby incorporated by reference.

The compositions, fusion proteins and polypeptides of the invention canbe administered to a subject on a presenting carrier. “Presentingcarrier,” as used herein, means any composition that presents thecompositions, fusion proteins and polypeptides of the invention to theimmune system of the subject to generate an immune response in thesubject. The presentation of the compositions, fusion proteins andpolypeptides of the invention would preferably include exposure ofantigenic portions of the viral protein to generate antibodies. Thecomponents (e.g., PAMP and a viral protein) of the compositions, fusionproteins and polypeptides of the invention are in close physicalproximity to one another on the presenting carrier. The compositions,fusion proteins and polypeptides of the invention can be attached to thepresenting carrier by covalent or noncovalent attachment. Preferably,the presenting carrier is biocompatible. “Biocompatible,” as usedherein, means that the presenting carrier does not generate an immuneresponse in the subject (e.g., the production of antibodies). Thepresenting carrier can be a biodegradable substrate presenting carrier,such as a polymer bead or a liposome. The presenting carrier can furtherinclude alum or other suitable adjuvants. The presenting carrier can bea virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g.,Salmonella) or a nucleic acid (e.g., plasmid DNA).

The compositions and methods of the invention can further include acarrier. “Carrier,” as used herein, refers to a molecule (e.g., protein,peptide) that can enhance stimulation of a protective immune response.Carriers can be physically attached (e.g., linked by recombinanttechnology, peptide synthesis, chemical conjugation or chemicalreaction) to a composition (e.g., a protein portion of a naturallyoccurring viral hemagglutinin) or admixed with the composition.

Carriers for use in the methods and compositions described herein caninclude, for example, at least one member selected from the groupconsisting of Tetanus toxoid (TT), Vibrio cholerae toxoid, Diphtheriatoxoid (DT), a cross-reactive mutant (CRM) of diphtheria toxoid, E. colienterotoxin, E. coli B subunit of heat labile enterotoxin (LTB), Tobaccomosaic virus (TMV) coat protein, protein Rabies virus (RV) envelopeprotein (glycoprotein), thyroglobulin (Thy), heat shock protein HSP 60Kda, Keyhole limpet hemocyamin (KLH), an early secreted antigentuberculosis-6 (ESAT-6), exotoxin A, choleragenoid, hepatitis B coreantigen, and the outer membrane protein complex of N. meningiditis(OMPC) (see, for example, Schneerson, R., et al., Prog Clin Biol Res47:77-94 (1980); Schneerson, R., et al., J Exp Med 152:361-76 (1980);Chu, C., et al., Infect Immun 40: 245-56 (1983); Anderson, P., InfectImmun 39:233-238 (1983); Anderson, P., et al., J Clin Invest 76:52-59(1985); Fenwick, B. W., et al., 54: 583-586 (1986); Que, J. U., et al.Infect Immun 56:2645-9 (1988); Que, J. U., et al. Infect Immun 56:2645-9(1988); (Que, J. U., et al. Infect Immun 56:2645-9 (1988); Murray, K.,et al., Biol Chem 380:277-283 (1999); Fingerut, E., et al., Vet ImmunolImmunopathol 112:253-263 (2006); and Granoff, D. M., et al., Vaccine11:Suppl 1:S46-51 (1993)).

Exemplary carrier proteins for use in the methods and compositionsdescribed herein can include at least one member selected from the groupconsisting of SEQ ID NOS: 275-282:

Cross-reactive mutant (CRM) of diphtheria toxin including, CRM197(SEQ ID NO: 275)GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSCoat protein of Tobacco mosaic virus (TMV) coat protein (SEQ ID NO: 276)MMAYSIPTPSQLVYFTENYADYIPFVNRLINARSNSFQTQSGRDELREILIKSQVSVVSPISRFPAEPAYYIYLRDPSISTVYTALLQSTDTRNRVIEVENSTNVTTAEQLNAVRRTDDASTAIHNNLEQLLSLLTNGTGVFNRTSFESASGLTWLVTTTPRTA Coat protein of alfalfa mosaic virus (AMV) (SEQ ID NO: 277)MSSSQKKAGGKAGKPTKRSQNYAALRKAQLPKPPALKVPVAKPTNTILPQTGCVWQSLGTPLSLSSSNGLGARFLYSFLKDFAAPRILEEDLIFRMVFSITPSHAGSFCLTDDVTTEDGRAVAHGNPMQEFPHGAFHANEKFGFELVFTAPTHAGMQNQNFKHSYAVALCLDFDALPEGSRNPSYRFNEVWVERKAFPRAGPLRSLITVGLFDDADDLDRQ Coat protein of Potato virus X (SEQ ID NO: 278)MTTPANTTQATGSTTSTTTKTAGATPATTSGLFTIPDGEFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTTTAEAVVT LPPP Porins from Neisseria sp, e.g.,class I outer membrane protein of Neisseria meningitides(SEQ ID NO: 279)MRKKLTALVLSALPLAAVADVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYWTTVNTGSATTTTFVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRDAFELFLLGSGSDQAKGTDPLKNHQVHRLTGGYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF Major fimbrial subunit protein type I (Fimbrillin) (SEQ ID NO: 280)MVLKTSNSNRAFGVGDDESKVAKLTVMVYNGEQQEAIKSAENATKVEDIKCSAGQRTLVVMANTGAMELVGKTLAEVKALTTELTAENQEAAGLIMTAEPKTIVLKAGKNYIGYSGTGEGNHIENDPLKIKRVHARMAFTEIKVQMSAAYDNIYTFVPEKIYGLIAKKQSNLFGATLVNADANYLTGSLTTFNGAYTPANYANVPWLSRNYVAPAADAPQGFYVLENDYSANGGTIHPTILCVYGKLQKNGADLAGADLAAAQAANWVDAEGKTYYPVLVNFNSNNYTYDSNYTPKNKIERNHKYDIKLTITGPGTNNPENPITESAHLNVQCTVAEWVLVGQNATWMycoplasma fermentans macrophage activating lipopeptide (MALP-2)(SEQ ID NO: 281)MKKSKKILLGLSPIAAVLPAVAVSCGNNDESNISFKEKDISKYTTTNANGKQVVKNAELLKLKPVLITDEGKIDDKSFNQSAFEALKAINKQTGIEINSVEPSSNFESAYNSALSAGHKIWVLNGFKHQQSIKQYIDAHREELERNQIKIIGIDFDIETEYKWFYSLQFNIKESAFTTGYAIASWLSEQDESKRVVASFGVGAFPGVTTFNEGFAKGILYYNQKHKSSKIYHTSPVKLDSGFTAGEKMNTVINNVLSSTPADVKYNPHVILSVAGPATFETVRLANKGQYVIGVDSDQGMIQDKDRILTSVLKHIKQAVYETLLDLILEKEEGYKPYVVKDKKADKKWSHFGTQKEKWIGVAENHFSNTEEQAKINNKIKEAIKMFKELPEDFVKYINSDKALKDGNKIDNVSERLEAIISAINKAAK  p19 protein of Mycobacterium tuberculosis (SEQ ID NO: 282)ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN 

The compositions of the invention can further include at least oneadjuvant. Adjuvants contain agents that can enhance the immune responseagainst substances that are poorly immunogenic on their own (see, forexample, Immunology Methods Manual, vol. 2, I. Lefkovits, ed., AcademicPress, San Diego, Calif., 1997, ch. 13). Immunology Methods Manual isavailable as a four volume set, (Product Code Z37, 435-0); on CD-ROM,(Product Code Z37, 436-9); or both, (Product Code Z37, 437-7). Adjuvantscan be, for example, mixtures of natural or synthetic compounds that,when administered with compositions of the invention, such as proteinsthat stimulate a protective immune response made by the methodsdescribed herein, further enhance the immune response to the protein.Compositions that further include adjuvants may further increase theprotective immune response stimulated by compositions of the inventionby, for example, stimulating a cellular and/or a humoral response (i.e.,protection from disease versus antibody production). Adjuvants can actby enhancing protein uptake and localization, extend or prolong proteinrelease, macrophage activation, and T and B cell stimulation. Adjuvantsfor use in the methods and compositions described herein can be mineralsalts, oil emulsions, mycobacterial products, saponins, syntheticproducts and cytokines Adjuvants can be physically attached (e.g.,linked by recombinant technology, by peptide synthesis or chemicalreaction) to a composition described herein or admixed with thecompositions described herein.

The dosage and frequency (single or multiple doses) administered to asubject can vary depending upon a variety of factors, including priorexposure to an antigen, a viral protein, the duration of viralinfection, prior treatment of the viral infection, the route ofadministration of the composition, fusion protein or polypeptide; size,age, sex, health, body weight, body mass index, and diet of the subject;nature and extent of symptoms of flavivirus exposure, flavivirusinfection and the particular flavivirus responsible for the infection(e.g., a West Nile flavivirus, a Dengue flavivirus, a Langat flavivirus,a Kunjin flavivirus, a Murray Valley encephalitis flavivirus, a Japaneseencephalitis flavivirus, a Tick-borne encephalitis flavivirus, a Yellowfever flavivirus and a hepatitis C flavivirus), kind of concurrenttreatment, complications from the flavivirus exposure, flavivirusinfection or other health-related problems. Other therapeutic regimensor agents can be used in conjunction with the methods and compositions,fusion proteins or polypeptides of the present invention. For example,the administration of the compositions, fusion proteins or polypeptidescan be accompanied by other viral therapeutics or use of agents to treatthe symptoms of the flavivirus infection (e.g., high fever, numbness,DHF, meningoencephalitis). Adjustment and manipulation of establisheddosages (e.g., frequency and duration) are well within the ability ofthose skilled in the art.

The teachings of all of the references cited herein are herebyincorporated by reference in their entirety.

The present invention is further illustrated by the following examples,which are not intended to be limited in any way.

EXEMPLIFICATION Example 1

Materials and Methods

PCR Amplification and DNA Primers

All PCR amplifications were performed using Pfu Ultra Hotstart PCRMaster Mix (Catalog number 600630) from Stratagene (La Jolla, Calif.)according to the manufacturer's recommendations. DNA primers werepurchased from Sigma Genosys and are described below.

STF28BGF-1: (SEQ ID NO: 41) CTCGGGAGATCTGCACAAGTAATCAACACTAACAGTCT STF28MCR-1: (SEQ ID NO: 42) CCATGGGCTAGCAGGATCCACCGGCGCTCCCTGCACGTTCA STF28MCF-2: (SEQ ID NO: 43) GGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCG STF28ECR-2: (SEQ ID NO: 44)TCTGCAGAATTCACGTAACAGAGACAGCACGTTCTGCGGGACGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTGCAGA  pET24AR: (SEQ ID NO: 45)5 TCCGGCGTAGAGGATCGAGA  STF2-E3R3: (SEQ ID NO: 46)CAATTGACCTTCAAGCTTCGAATTGCCCTTACGTAACAGAGACAGCACG TTCTG AX-E3F3:(SEQ ID NO: 47) AAGCTTGAAGGTCAATTGGAATTCCCTAGGACTAGTATGGAAAAATTGCAGTTGAAG  pET24AF:  (SEQ ID NO: 48) GCTTAATGCGCCGCTACAGG  5′WNE28: (SEQ ID NO: 49) GCGGCCGCTCATGGAAAAATTGCAGTTGAAGGGAACAACC  3′WNE28: (SEQ ID NO: 50) CCGCGGTTTGCCAATGCTGCTTCCAGACTTGT  NdeI-STF2: (SEQ ID NO: 51) CCGGCATGCCATATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCBlpI-EdIII:  (SEQ ID NO: 52)GCATGCTCAGCTTATTAAGGGTTTGCCAATGCTGCTTCCCAGACTTGTG JE EIII primer: (SEQ ID NO: 53) TACGTGAATTCAGCAGATATCCAGCAC Cloning of pET/STF2Δ.EIII

Full length flagellin of Salmonella typhimurium fljb (flagellin phase 2)(also referred to herein as “STF2”) is encoded by a 1.5 kb gene. Atruncated version of the STF2 (STF2Δ, SEQ ID NO: 3, encoded by SEQ IDNO: 4) was generated by deleting the hyper-variable region that spansamino acids 170 to 415 of SEQ ID NO: 1. The deleted region was replacedwith a short flexible linker (GAPVDPASPW, SEQ ID NO: 56) designed tofacilitate interactions of the NH2 and COOH termini sequences necessaryfor TLR5 signaling. To generate this construct, a two-step PCR was used.In the first reaction, STF2.OVA ((FIG. 61) SEQ ID NO: 152 encoding aminoacid sequence SEQ ID NO: 153 of FIG. 62) served as the DNA template andSTF28BGF-1 and STF28MCR-1 were used as primer pairs. In a separatereaction, the same DNA template was combined with primers STF28MCF-2 andSTF28ECR-2.

The PCR amplification reactions generated about 500 bp and about 270 bpfragments, respectively. These PCR products were combined in a final PCRreaction using STF28BGF-1 and STF28ECR-2 as primers. The amplified DNAproduct from this reaction (about 0.77 kb) was digested with BglII andEcoRI restriction enzymes and ligated into pMTBiP/V5-His B (Invitrogen,Carlsbad, Calif.) that had previously been digested with BglII and EcoRIand treated with alkaline phosphatase. An aliquot of the ligation mixwas used to transform TOP10 cells (InVitrogen, Carlsbad, Calif.). PCRscreening was performed using vector specific primers, pMTFOR(methionine promoter) (CATCTCAGTGCAACTAAA, SEQ ID NO: 156) and BGHREV(bovine growth hormone poly A) (TAGAAGGCACAGTCGAGG, SEQ ID NO: 157), toidentify several positive clones. All positive clones were furtheranalyzed by restriction mapping analysis and confirmed by DNAsequencing. The resultant construct pMT/STF2Δ was used to generatepMT/STF2Δ.EIII+.

The domain III of the West Nile virus envelope protein (FIGS. 45 and 46)of pET/STF2Δ.EIII+ (SEQ ID NOS: 70, 71) was derived from the Drosophilaexpression plasmid pMT/STF2.E. This plasmid contains full-length STF2(amino acids 1-506, SEQ ID NO: 1) fused to the West Nile Virus envelopeprotein (amino acids 1-406, SEQ ID NO:39, FIG. 45). The pMT/STF2.E (SEQID NO: 158) clone AX-1 was used as a DNA template and 5′WNE28 (SEQ IDNO: 49) and 3′WNE28 (SEQ ID NO: 50) served as primers for PCRamplification. In order to facilitate restriction analysis andsubsequent cloning steps, the 5′ primer encoded a novel Nod site (NewEngland Biolabls, Beverly, Mass.) and the 3′ primer contained a uniqueSacII site. The amplified EIII+ DNA fragment (345 bp; SEQ ID NO: 178that encodes amino acids 292-406 of SEQ ID NO: 39) was subcloned intopCR-Blunt II-TOPO cloning vector (InVitrogen, Carlsbad, Calif.) togenerate plasmid TOPOEIII. A stop codon was subsequently introduceddownstream of the EIII+ sequence by blunting the SacII and SpeIrestriction sites using T4 DNA polymerase.

To generate pMT/STF2Δ.EIII+ (SEQ ID NOS: 70, 71), the EIII+ fragment wasisolated from TOPOEIII+ using Nod and BamHI restriction sites andligated into the Nod and SacII restriction sites in pMT/STF2Δ. The BamHIsite of the EIII+ DNA fragment and the SacII site of pMTSTF2Δ wereblunted with T4 DNA polymerase prior to ligation. The STF2Δ.EIII+sequence (SEQ ID NOS: 70, 71) from pMT/STF2Δ.EIII+ was isolated by PCRamplification using the primers NdeI-STF2 and BlpI-EdIII. To generatepET/STF2Δ.EIII+ (SEQ ID NO: 71), the PCR product was digested with NdeIand BlpI and ligated into pET24a plasmid that had been predigested withNdeI and BlpI. The ligation mix was transformed into Mach-1 cells(InVitrogen, Carlsbad, Calif.) and the cells were grown on LBsupplemented with 50 μg/ml kanamycin. Several colonies were screened byrestriction mapping and were verified by DNA sequencing.

Cloning of pET/STF2.EIII+

The West Nile virus EIII+ sequence of pET/STF2.EIII+ (SEQ ID NOS: 54,55) was derived from pETSTF2.E (SEQ ID NOS: 158, 159). This E. coliexpression plasmid contains full-length STF2 (amino acids 1-506) fusedto the West Nile Virus envelope protein (amino acids 292-406 of SEQ IDNO: 39, which is SEQ ID NO: 7). In two independent PCR reactions,pET/STF2.E was used as the DNA template. One reaction used the primerspET24AR:5 (SEQ ID NO: 45) and STF2-E3R3: (SEQ ID NO: 46) and the otherused AX-E3F3 (SEQ ID NO: 47) and pET24AF (SEQ ID NO: 48). These PCRreactions generated a 1.5 kb fragment that consisted of full-length STF2and a 340 bp fragment that comprised the EIII domain plus additionalamino acids that extended into domain I of the envelope protein.Aliquots of these PCR amplification reactions were combined, and the twoproducts served as templates for a PCR reaction with the externalprimers pET24AR (SEQ ID NO: 45) and pET24AF (SEQ ID NO: 48). Thisresulted in the generation of about a 1.8 kb DNA fragment that fusedEIII+ sequence (SEQ ID NO: 178, a nucleic acid sequence encoding aminoacids 292-406 of SEQ ID NO: 39, which is SEQ ID NO: 7) to STF2. The PCRproduct was digested with NdeI and BlpI and gel purified and ligated bycompatible ends to a pET24a vector that had previously been digestedwith compatible enzymes and de-phosphorylated. The ligation mix wastransformed into Mach-1 cells (InVitrogen, Carlsbad, Calif.) asdescribed for pET/STF2Δ.EIII+. Several colonies were screened byrestriction mapping and two clones were verified by DNA sequencing.

Cloning of pET/STF2Δ.JEIII+

A portion of the envelope protein of a Japanese encephalitis virus (JEV)(strain SA-14-14-2 (Jai, L., et al., Chin Med J (Eng) 116:941-943(2003)); currently employed in a JEV vaccine encoded by domain III wascustom synthesized by DNA 2. Inc (Menlo Park, Calif.). The portion ofdomain III was excised from the pJ2:G01510 using NotI and Blp I sitethat flank the insert. The DNA insert was gel isolated and cloned bycompatible ends to pET24A/STF2Δ.EIII+ (SEQ ID NOS: 70, 71) that hadpreviously been digested with the appropriate enzymes to release theWest Nile virus EIII+ insert. The deleted vector was then gel purifiedand ligated to an aliquot of JE EIII+. The ligation mix was used totransform TOP-10 cells (InVitrogen, Carlsbad, Calif.) and the cells weregrown on LB supplemented with 50 μg/ml kanamycin. Several colonies werescreened by restriction mapping and were verified by DNA sequencing.

The resulting construct, pET24A/STF2Δ.JEIII (SEQ ID NOS: 5, 6) was BLR(DE3) strain (Novagen) and expression was monitored in several clonesusing Commassie Blue staining which was confirmed by Western blot usinganti-flagellin antibodies. Using, pET24A/STF2Δ.JEIII+ as the DNAtemplate and the JE EIII+ oligonucleotide as primer (SEQ ID NO: 53) thecysteine residue in the linker region between STF2Δ and JEIII+ waschanged to a serine residue using QuikChange Site Directed MutagenesisKit (Stratagene, LaJolla, Calif.) according to the manufacturer'sinstructions. The clone was verified by sequencing and assayed forexpression as described for pET24A/STF2Δ.JEIII+ above.

When a cysteine residue in a linker in change to a serine residue thefusion protein in also referred to herein by inclusion of an “s” in thedesignation of the fusion protein. For example, “STF2Δ.EIII+” includes acysteine residue in the linker (FIG. 29, SEQ ID NO: 71), whereas“STF2Δ.EIIIs+” include a serine residue substituted for the cysteineresidue in the linker (FIG. 30, SEQ ID NO: 72).

Cloning the EIII Domain of Each Dengue Virus Fused to the C-Terminal Endof Flagellin (STF2Δ)

Initially, obtaining biologically active material from the fusion of theentire envelope protein of West Nile virus was difficult, perhaps due tothe presence of multiple cysteines residues (12 cysteines) in theenvelope protein (see SEQ ID NO: 39, FIG. 45). However, when the regionencoding domain III (EIII) of the protein was sub-cloned, the fusionprotein was abundantly expressed in E. coli and was highly efficaciousin mice. Although there is an overall sequence dissimilarity among the 4distinct DEN viruses (Den1, Den2, Den3, Den4, SEQ ID NOS: 160-167, FIGS.67-74) the three-dimensional structures within domain III of theenvelope protein are similar among the flaviviruses. This domain in DENand other flaviviruses encodes the majority of the type-specificcontiguous critical/dominant neutralizing epitopes. Domain III of thedengue viruses (Den1, Den2, Den3 and Den4) has been expressed inbacteria and shown to be immunogenic, capable of inducing neutralizingantibodies in experimental animals (Simmons, M., et al., Am. J. Trop.Med. Hyg 65:159 (2001)). Domain III corresponding to residues about 295to about 399 (exact numbering depends on the particular DEN virus, forexample, of SEQ ID NOS: 160, 162, 164, 166) of the four different DENviruses have been codon-optimized for expression in E. coli. Thesynthetic gene was amplified by using PCR and sub-cloned into the NotIsite of the vector pET/STF2Δ generating pET/STF2Δ.DEN1EIII,pET/STF2Δ.DEN2EIII, pET/STF2Δ.DEN3EIII and pET/STF2Δ.DEN4EIII (SEQ IDNOS: 80, 82, 84 AND 86).

E. coli Production of STF2.EIII+, STF2Δ.EIII+, STF2Δ.EIIIs+ andSTF2Δ.JEIII+

Cell cultures (6 L) of BLR(DE3) pLysS that harbor pETSTF2.EIII+ (SEQ IDNOS: 54, 55), pETSTF2Δ.EIII+ (SEQ ID NOS: 70, 71), pETSTF2Δ.EIIIs+(SEQID NOS: 72, 73) or pETSTF2Δ.JEIII+ SEQ ID NOS: 5, 6) were grown in LBmedium containing 15 μg/ml kanamycin, 12.5 μg/ml tetracycline and 24μg/ml chloramphenicol. At an OD₆₀₀ of about 0.6 protein expression wasinduced with 1 mM IPTG for about 3 h at about 37° C. Followinginduction, cells were harvested by centrifugation (7000 rpm×7 minutes ina Sorvall RC5C centrifuge) and resuspended in 2×PBS, 1% glycerol, DNAse,1 mM PMSF, protease inhibitor cocktail and 1 mg/ml lysozyme. Thesuspension was passed through a microfluidizer to lyse the cells and thelysate was centrifuged (45,000 g for one hour in a Beckman Optima Lultracentrifuge) to separate the soluble fraction from inclusion bodies.Under these growth and induction conditions, STF2.EIII+ was expressed asa soluble protein and STF2Δ.EIII+ (SEQ ID NOS: 70, 71), STF2Δ.EIIIs+(SEQ ID NOS: 72, 73) and STF2Δ.JEIII+ (SEQ ID NOS: 5, 6) formedinclusion bodies.

Purification of STF2.EIII+

Cell lysate containing soluble STF2.EIII+ (SEQ ID NOS: 54, 55) wasapplied to Sepharose Q resin (Amersham Biosciences, Piscataway, N.J.) inthe presence of 0.5 M NaCl to reduce DNA, endotoxin, and othercontaminants. The flow-through fraction was collected and theconductivity adjusted by a 10-fold dilution with buffer A (Buffer A: 100mM Tris-C1, pH 8.0). The diluted material was re-loaded onto Q Sepharoseand bound protein was eluted with a linear gradient from 20% to 60%Buffer B (Buffer B: 100 mM Tris-Cl, 1 M NaCl, pH 8.0). Fractionscontaining STF2.EIII+ were pooled and further processed by Superdex-200gel (SD200) filtration chromatography in the presence of Na-deoxycholateto remove residual endotoxin (running buffer: 1% Na-deoxycholate, 100 mMNaCl, 100 mM Tris-HCl, 1% glycerol, pH 8.0). Following SD200chromatography, the eluted protein was loaded directly onto Q Sepharoseand washed extensively with buffer A to remove detergent. Bound proteinwas again eluted with a linear gradient from 20% to 60% Buffer B. In onepreparation (Batch 057), this step was substituted with a detergentremoval procedure using Extract-D detergent removal gel (PierceBiotechnology, Rockford, Ill.). The purified protein was dialyzedagainst buffer containing 50 mM Tris, 100 mM NaCl and 1% glycerol andstored at −80° C.

Purification of STF2Δ.EIII+

STF2Δ.EIII+ inclusion bodies were collected by low-speed centrifugation(7000 rpm×7 minutes in a Sorvall RC5C centrifuge) and solubilized withbuffer containing 8 M urea, 100 mM Tris-HCl, 5 mM EDTA, pH 8.0. The ureaconcentration of the solubilized protein was adjusted to 1 M and thesample was loaded onto Q Sepharose. The bound protein was eluted using alinear gradient from 0% to 100% Buffer B. (Buffer A: 100 mM Tris-HCl, 5mM EDTA, 1 M urea, pH 8.0. Buffer B: 100 mM Tris-C1, 5 mM EDTA, 1 MNaCl, 1 M urea, pH 8.0). Due to the formation of protein aggregatesfollowing elution, the urea concentration of the Q Sepharose materialwas adjusted to 8 M. The protein was further purified by gel filtrationchromatography using SD200. The column was pre-equilibrated with 100 mMTris-HCl, pH 8.0, 100 mM NaCl, 1% glycerol, 8 M urea plus 1%Na-deoxycholate. The eluted protein was subjected to a second IEXchromatography step using Source Q to remove 1% Na-deoxycholate. Boundprotein was eluted with a linear gradient from 20% to 60% Buffer B.(Buffer A: 100 mM Tris-C1, pH 8.0, 8 M urea, 5 mM EDTA. Buffer B: 100 mMTris-HCl, pH 8.0, 5 mM EDTA, 8 M urea, 1 M NaCl). Final polishing of theprotein was completed by gel filtration chromatography using SD200(Running Buffer: 100 mM Tris-HCl, pH 8.0, 8 M urea, 100 mM NaCl and 1%glycerol). Reducing agent was added to the SD200 fraction (2.5 mM DTT)and the protein was refolded by step-wise dialysis against decreasingconcentrations of urea. The urea concentration was reduced sequentiallyagainst buffers that contained 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1%glycerol and 6 M, 4 M, 2 M or no urea.

Refolding and Purification of STF2Δ.EIII+ Trimer

STF2Δ.EIII+ (SEQ ID NOS: 70, 71) from urea-solubilized inclusion bodieswas efficiently refolded to form trimer product by simple dialysis asdescribed above the trimer (3 of the STFΔ.EIII fusion proteins) wasdeduced based on molecular weight in SDS-PAGE. Following dialysis,endotoxin was removed by multiple extractions with Triton X-114. Thetrimer was purified and separated from monomer and aggregates by 5200size exclusion chromatography. The final product migrated as a singleband with an apparent molecular weight of about 130 kDa on SDS-PAGE.

Refolding and Purification of STF2Δ.EIII+ Monomer

The monomeric form of STF2Δ.EIII+ (SEQ ID NOS: 70, 71) was producedconsistently and efficiently by refolding using rapid dilution, whichprevented individual STF2Δ.EIII+ fusion proteins from interacting withone another to form meutimers, such as trimers (supra). STF2Δ.EIII+solubilized from inclusion bodies in 4M urea was raised to 8M ureawithout reductant. The protein was then rapidly diluted inTris/NaCl/glycerol buffer, pH 8.0, to about 0.1 mg/ml and a final ureaconcentration of 0.1M at room temperature. The monomer was furtherpurified and separated from aggregates by S200 size exclusionchromatography. The final product migrated as a single band with anapparent molecular weight of about 43 kDa on SDS-PAGE.

Purification of STF2Δ.EIIIs+(Serine Substitution of the Linker BetweenSTF2Δ and EIII+, SEQ ID NO: 72)

STF2Δ.EIIIs+ (SEQ ID NOS: 72, 73) from solubilized inclusion bodies wasrefolded using a rapid dilution method similar to that used to refoldthe STF2Δ.EIII+ monomer. The refolded protein was captured on a butylsepharose column and eluted while removing most of the endotoxincontamination. Eluate from the butyl sepharose purification wasconcentrated and put through 4 cycles of Triton X-114 extractions toreduce endotoxin levels down to about <0.1 EU/μg before a finalpurification step over SD200 gel filtration. The final pooled productmigrated as a single band with an apparent molecular weight of about 43kDa on SDS-PAGE and contained a trace amount of Triton X-114 (about0.000015%).

Purification of STF2Δ.JEIII+ (SEQ ID NOS: 5, 6)

Protein was isolated from inclusion bodies under denaturing conditions.Inclusion bodies were washed with detergent (0.5% Triton X 100) andsolubilized in 8 M urea, resulting in partial purification of the targetprotein. For endotoxin removal, protein was applied on a Source S cationexchange column at low pH (about 3.5) and eluted with a salt gradient (0to about 1M NaCl). The protein was refolded using rapid dilution asdescribed for STF2Δ.EIII+ monomer. The protein was then concentrated andfurther purified using SD200 to separate the monomeric form of theprotein from aggregates. The purified material migrated with an apparentmolecular weight of about 43 kDa on SDS PAGE and contained acceptablelevels of endotoxin (about 0.03 EU/ug).

Fed Batch Production of Fusion Proteins

STF2Δ.EIIIs+ was produced in an aerobic bioreactor using a fed batchprocess. Three control loops were placed to control pH by acid (2 N HCl)or base (3 N NH₄OH) addition, temperature by heating (heating blanket)or cooling (time cycled cooling loop), and dissolved oxygen bycompressed air flow (manually controlled), agitation (mixing speed) andO₂ flow (timed cycled) in cascade. Cells [BLR(DE3) pLysS that harbor theSTF2Δ.EIIIs+ were adapted to and banked in MRSF media (see infra), andfrozen in 25% glycerol. Cells were scaled up for the bioreactor byadding 1 mL of banked cells to 1 L of MRSF media and agitating at about37° C. for about 15.5 to about 16.5 hours. Cells from the scale upprocess were added in a about 1:10 ratio to MRSF or MRBR synthetic mediaat about 37° C. and about 0.5 vvm air flow.

The process was run in batch mode at about 37° C. until the cells oxygenconsumption was such that the compressed air flow is about 1.5 vvm andthe agitation is at the maximum, about 6 hours, when the temperature isdropped to between about 25° C. and about 33° C. The feed can be startedbefore the culture is induced, or up to about 1 and about ½ hours after.The feed rate can be kept constant, or adjusted based on processvariables (dissolved oxygen, glucose concentration). The culture wasinduced with IPTG upon batch glucose exhaustion. The culture wasmaintained for a minimum of about 2 hours and a maximum of about 20hours.

MRBR Media Trace Metal Solution 1000x Composition g/L Component g/LGlucose 20 EDTA, disodium 5 KH₂PO₄ 2.2 FeSO₄(7H₂O) 10 (NH₄)₂SO₄ 4.5ZnSO₄(7H₂O) 2 Citric Acid 1.0 MnSO₄(H₂O) 2 MgSO₄(7H₂0) 1.0 CoC1₂(6H₂O)0.2 CaCl₂ 0.04 CuSO₄(5H₂O) 0.1 Trace Metals 1 ml Na₂MoO₄(2H₂O) 0.2Thiamine HCl 0.01 H₃BO₃ 0.1 Antifoam 0.05 MRSF Media Feed MediaComposition g/L Composition g/L Glucose 10 (20 in bioreactor) NaC1 0.5KH₂PO₄ 7.8 FeSO₄(7H₂O) 2 (NH₄)₂SO4 2.33 CaC1₂ 3.5 Citric Acid 1.0MgSO₄(7H₂O) 12 MgSO₄(7H₂0) 1.0 Thiamine HC1 1 CaCl₂ 0.04 Trace Metals 1ml Trace Metals 1 ml Glucose 100 Thiamine HCl 0.01 Kanamycin 0.0075(shake flask only)

STF2Δ.EIIIs+ was produced as inclusion bodies. Upon harvest, the cellswere separated from the conditioned media by centrifugation (BeckmanAvanti J-20 XP, JLA 8.1000 rotor, 10 kxg for about 20 minutes at about4° C.) and resuspended in equal volume of 50 mM Tris, 100 mM NaCl, 1 mMEDTA, pH 8.0. The centrifugation was repeated under the same conditions,with the cells resuspended in a minimum volume of the same buffer. Thesuspension was passed through a homogenizer (APV-1000) at >10,000 psifor at least two passes.

The solids can be separated and the STF2Δ.EIIIs solubilized by one ofthree methods; centrifugation, filtration, or fluidized bedchromatography.

Method 1

Solids are separated by centrifugation (Beckman Avanti J-20 XP, JA 20rotor, 20 kxg for 20 minutes at 4° C.) and resuspended in 50 mM tris, 1m NaCl, 1 mM EDTA, 1% glycerol, 0.5% Triton X-100, pH 8.0. This processwas repeated up to 6 times (total) at increasing speeds and times (to amaximum of about 40 kxg for about 20 minutes). After the final pelletrecovery, the pellet was resuspended in 50 mM Tris, 0.1M NaCl, 1 mMEDTA, pH 8.0 and clarified by centrifugation (Beckman Avanti J-20 XP, JA20 rotor, 40 kxg for about 20 minutes at about 4° C.) The pellet wasresuspended and dissolved in 50 mM Tris, 0.1M NaCl, 1 mM EDTA, 4 M urea,pH 8.0. Insolubles were removed by centrifugation (Beckman Avanti J-20XP, JA 20 rotor, 40 kxg for about 50 minutes at about 4° C.), thesupernatant retained for further processing.

After the multiple washes described above, STF2Δ.EIIIs can also bedissolved in 50 mM acetate, 10 mM NaCl, 8M urea, pH about 4.1 to about5.3 and clarified by centrifugation (Beckman Avanti J-20 XP, JA 20rotor, 20 kxg for about 20 minutes).

Method 2

After homogenization, the lysate was captured in body feed andSTF2Δ.EIIIs+ extracted with urea containing buffer. Body feed is afilter aid designed to trap particles in a cake above a depth filter.The body feed (Advanced Minerals Corporation CelPure 65) is a diatomite(silica powder) with a high surface area and low permeability, retaining<0.2 μm particles. The filter aid was pre-mixed with the lysate andpumped over a depth filter (Ertel 703), building up a cake containingboth body feed and lysate particles. The suspension creates a depthfilter as the particles settle on the filter pad. A 50 mM Tris, 100 mMNaCl pH 8.0 wash was performed to remove soluble proteins and nucleicacids. A subsequent wash with 50 mM Tris, 100 mM NaCl, 4 M urea, pH 8solubilizes and removes the STF2Δ.EIIIs from the body feed for furtherprocessing.

Method 3

After the cells were initially resuspended in buffer, they wereresuspended in sodium chloride and urea containing buffer at pH about 6to about 8 and homogenized. The lysate was applied on a Streamline CSTfluidized bed column (GE Healthcare) where the STF2Δ.EIIIs+ binds to theresin and the particulates flow through. STF2Δ.EIIIs+ may be eluted inlow salt conditions at a pH greater than the load pH, in the presence orabsence of detergents such as Triton X-100 or polysorbate 80.

SDS-PAGE

Proteins (typically about 5 μg) were diluted in SDS-PAGE sample bufferwith and without β-mercaptoethanol. The samples were boiled for 5minutes and loaded onto a 4-20% SDS polyacrylamide gel. Followingelectrophoresis, gels were stained with coomassie blue to visualizeprotein bands.

Endotoxin Assay

Endotoxin levels were measured using the QCL-1000 QuantitativeChromogenic LAL test kit (BioWhittaker #50-648U, Walkersville, Md.),following the manufacturer's instructions for the microplate method.

Protein Assay

Protein concentrations were determined by the MicroBCA Protein AssayReagent Kit in a 96-well format using BSA as a standard (PierceBiotechnology, Rockford, Ill.).

TLR5 Bioactivity Assay

HEK293 cells (ATCC, Catalog No. CRL-1573 Manassas, Va.) constitutivelyexpress TLR5, and secrete several soluble factors, including IL-8, inresponse to TLR5 signaling. Cells were seeded in 96-well microplates(about 50,000 cells/well), fusion proteins added and incubatedovernight. The next day, the conditioned medium was harvested,transferred to a clean 96-well microplate, and frozen at −20° C. Afterthawing, the conditioned medium was assayed for the presence of IL-8 ina sandwich ELISA using an anti-human IL-8 matched antibody pair (Pierce,#M801E and #M802B, Rockford, Ill.) following the manufacturer'sinstructions. Optical density was measured using a microplatespectrophotometer (FARCyte, Amersham Biosciences, Piscataway, N.J.).

Plaque Reduction Neturalization Test (PRNT)

PRNT was performed according to Wang, et al., J. Immunol. 167:5273-5277(2001). Briefly, serum samples were heat inactivated by incubation in a56° C. water bath for about 30 min and were serially diluted in PBS with5% gelatin from 1/10 to 1/2560. West Nile virus was diluted in PBS with5% gelatin so that the final concentration was about 100 PFU/well. Viruswas mixed with about 75 μA serum in a 96-well plate at about 37° C. forabout 1 h. Aliquots of serum-virus mixture were inoculated ontoconfluent monolayers of Vero cells in a six-well tissue culture plate.The cells were incubated at about 37° C. for 1 h, and the plates wereshaken every 15 min. The agarose overlay was then added. The overlay wasprepared by mixing equal volumes of a solution consisting of 100 ml2×MEM (Life Technologies) with sterile 2% agarose. Both solutions wereplaced in a 40° C. water bath for 1 h before adding the overlay. Thecells were incubated for 4 days at 37° C. in a humidified 5% CO₂-airmixture. A second overlay with an additional 4% neutral red was added onday 5. Virus plaques were counted about 12 h later.

Antigenicity of STF2Δ-Fusion Proteins

ELISA plates (96-well) were coated overnight at 4° C. with serialdilutions (100 μl/well) of purified STF2Δ-fusion proteins (SEQ ID NOS:158, 159, 54, 55, 70, 71) in PBS (about 2 μg/ml). Plates were blockedwith 200 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen) for onehour at room temperature. The plates were washed 3× in PBS-Tween, andthen incubated with antibodies reactive with flagellin or the E domainof the construct. The expression of flagellin was detected using the mAb6H11 (Intotek), while the antigenicity of WNV-E was monitored using apanel of mAb (5C5, 7H2, 5H10, 3A3, and 3D9) (Beasley, D. W., et al., J.Virol. 76:13097-13100 (2002)) were purchased from Bioreliance (RoadRockville, Md.). Antibodies diluted in ADB (about 100 μd/well) wereincubated overnight at 4° C. The plates were washed 3× with PBS-T.HRP-labeled goat anti-mouse IgG antibodies (Jackson Immunochemical, WestGrove, Pa.) diluted in ADB were added (100 μl/well) and the plates wereincubated at room temperature for 1 hour. The plates were washed 3× withPBS-T. After adding TMB (3,3′,5,5′-tetramentylbenzidine) Ultra substrate(Pierce Biotechnology, Rockford, Ill.) and monitoring color development,A₄₅₀ was measured on a Tecan Farcyte microspectrophotometer.

Immunization of Mice

C3H/HeN mice (10 per group) were immunized intraperitoneally orsubcutaneously with the indicated concentrations of fusion proteins orsynthetic peptides on days 0, 14 and 28. On days 21 and 35, immunizedanimals were bled by retro-orbital puncture. Sera were harvested byclotting and centrifugation of the heparin-free blood samples. On day35, mice were challenged with a lethal dose of WNV strain 2741 (Wang,T., et al., J. Immunol. 167:5273-5277 (2001)). Survival was monitoredfor 21 days post-challenge.

Serum Antibody Determination

West Nile envelope protein specific IgG levels were determined by ELISA.ELISA plates (96-wells) were coated overnight at about 4° C. with 100μl/well of West Nile E protein mAb 5C5, 7H2, 5H10, 3A3, and 3D9(Beasley, D. W., et al., J. Viro. 76:13097-13100 (2002)) (Bioreliance,Road Rockville, Md.) in PBS at a concentration of 2 μg/ml. Plates wereblocked with 200 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen,San Diego Calif.) for one hour at room temperature. The plates werewashed 3× in PBS-T. Dilutions of the sera in ADB were added (100μl/well) and the plates were incubated overnight at 4° C. The plateswere washed 3× with PBS-T. HRP-labeled goat anti-mouse IgG antibodies(Jackson Immunochemical, West Grove, Pa.) diluted in ADB were added (100μl/well) and the plates were incubated at room temperature for 1 hour.The plates were washed 3× with PBS-T. After adding TMB(3,3′,5,5′-tetramentylbenzidine) Ultra substrate (Pierce Biotechnology,Rockford, Ill.) and monitoring color development, A₄₅₀ was measured on aTecan Farcyte microspectrophotometer.

Production of Pam3Cys.WNV001 Peptide Synthesis

Pam3Cys.WNV001 was synthesized by Bachem Bioscience, Inc. (King ofPrussia, Pa.). WNV001 is a 20 amino acid peptide (SEQ ID NO: 168) of theWest Nile virus envelope protein chemically coupled to atri-palmitoylcysteine (Pam3Cys) moiety through the amino terminal serineresidue of the peptide. The chemical name for Pam3Cys.WNV001 is[Palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-LTSGHLKCRVKMEKLQLKGT(SEQ ID NO: 168) acetate salt]. The molecular mass of Pam3Cys.WNV001 is3163.3 daltons. The peptide was synthesized by Bachem using solid phasesynthesis methodologies and FMOC chemistry. The amino acid sequence ofPam3Cys.WNV001 was assembled on an H-Pro-2-chlorotrityl chloride resinby solid phase peptide synthesis. The peptide chain was elongated bysuccessive coupling of the amino acid derivatives. Each coupling stepwas preceded by an Fmoc-deprotection step and were accompanied byrepeated washing of the resin. After coupling of the last amino acidderivative, the final Fmoc-deprotection step was performed. Finally, thepeptide resin was washed and dried under reduced pressure. During solidphase peptide synthesis color indicator tests were performed for eachstep to monitor the completion of the Fmoc-cleavage and the subsequentcoupling of the amino acid derivatives. To couple Pam3Cys-OH to theelongated peptide, the lipid moiety was pre-activated withN,N′-dicyclohexyl-carbodiimide (DCCI) in the presence of1-hydroxybenzotriazole (HOBt). The resulting solution was filtered andadded to the peptide resin. At the end of the reaction time the peptideresin was washed and dried under reduced pressure. Color indicator testswere performed to control the coupling of Pam3Cys-OH. The completedpeptide was cleaved from the resin by incubating with trifluoroaceticacid (TFA). The liberated product (crude peptide material) wasprecipitated from the reaction mixture and lyophilized. The crudeproduct was used for initial immunogenicity studies.

Synthesis of WNV-E Peptide Arrays

Peptide arrays (FIGS. 57 and 60) were synthesized by Sigma Genosys(Woodlands, Tex.).

Results:

West Nile Fusion Protein

West Nile virus (WNV) has emerged in recent years in temperate regionsof Europe and North America, presenting a threat to public and animalhealth. The most serious manifestation of WNV infection is fatalencephalitis (inflammation of the brain) in humans and horses, as wellas mortality in certain domestic and wild birds. WNV has also been asignificant cause of human illness in the United States. The envelopeglycoprotein of West Nile (WNV-E) and other flaviviruses may generateneutralizing and protective antibodies. By linking this antigen to aToll-like receptor ligand, the compositions, fusion proteins andpolypeptides described herein may target appropriate antigen presentingcells without the need for adjuvant or other immune modulatorformulations.

As described herein, several strategies have been implemented tofacilitate production of West Nile virus envelope (WNV-E) fusionproteins in E. coli. One approach is to engineer a smaller WNV-E antigenby fusing domain III (EIII) and, optionally, with amino acids of domainII of the WNV-E protein to full-length STF2 (e.g., STF2.E, STF2.EIII+).Domain III is responsible for virus-host interactions and retains manyWest Nile virus neutralizing antibody epitopes. It also contains only 2of the 12 cysteine residues present within the full length envelopeprotein, making expression in E. coli more feasible. A second approachhas been to delete the hyper-variable hinge region of flagellin (e.g.,STF2Δ) thereby creating a smaller fusion protein (STF2Δ.EIII+). Thehyper-variable region of flagellin is not required for TLR5 signalingand its removal may also reduce the immunogenic potential of flagellin.Both STF2.EIII+ and STF2Δ.EIII+ have been expressed in E. coli andpurified. The purified proteins have been characterized for TLR5signaling activity in bioassays and for E epitope display in ELISAassays using a panel of WNV-E polyclonal and neutralizing monoclonalantibodies. Results from these studies indicate that STF2Δ.EIII+ hashigher PAMP activity and more conformation-sensitive neutralizing WNV-Eepitopes than STF2.EIII+.

Purity of STF2.EIII+ and STF2Δ.EIII+

Several lots of STF2.EIII+ and STF2Δ.EIII+ have been produced in E. coliand purified (Table 1). STF2.EIII+ was expressed as a soluble proteinand purified under non-denaturing conditions using a 4-step process, asdescribed above, that included anion exchange chromatography and gelfiltration. Final yields from 6 L cultures ranged from about 0.9 mg toabout 3.8 mg and all preparations contained low levels of endotoxin asmeasured by standard LAL procedures (about <0.1 EU/μg protein, seesupra). In contrast, STF2Δ.EIII+ formed inclusion bodies in E. coli, andwas purified under denaturing conditions. All chromatography steps usedto purify STF2Δ.EIII+ required the use of 8M urea. Followingpurification, the denatured protein was refolded by step-wise dialysisto allow for gradual urea removal. Refolding was typically carried outat protein concentrations of about 0.3 mg/ml without any loss due toprotein precipitation. Two preparations of STF2Δ.EIII+ from a single 6 Lculture yielded about 1.2 and about 6.7 mg of protein, both of which hadacceptable endotoxin levels. As expected, purified STF2.EIII+ andSTF2Δ.EIII+ migrated on SDS PAGE under reducing conditions as about 65kDa and about 43 kDa proteins, respectively. Notably, STF2Δ.EIII+migrated slightly faster under non-reducing conditions. This alteredmigration may be due to disulfide bond formation involving the twocysteines residues in domain III of the envelope protein. As well, alarger species of STF2Δ.EIII+ was detected by Western blot analysiswhose molecular weight is consistent with a trimer form of the protein(“(STF2Δ.EIII+)_(x3) or 3 units of STF2Δ.EIII+”).

TABLE 1 Endotoxin levels and TLR-5 activity for STF2.EIII+ (SEQ ID NO:55) and STF2Δ.EIII+ (SEQ ID NO: 71) fusion proteins. Batch EndotoxinNumber Protein Yield (mg) Levels (EU/μg) TLR-5 EC₅₀ 052 STF2.EIII+ 3.80.03 >5000.00 ng/ml 054 STF2.EIII+ 0.9 0.02 1195.00 ng/ml 057 STF2.EIII+1.6 0.07 197.92 ng/ml 044 STF2Δ.EIII+ 1.2 0.07 1.13 ng/ml 045STF2Δ.EIII+ 6.7 0.07 4.34 ng/mlTLR5 Activity in the HEK293 IL-8 Assay

To compare the PAMP activity of both fusion proteins, a TLR5 bioassaywas performed. HEK293 IL-8 cells were treated with serial dilutions oftwo independent protein batches (FIGS. 47A and 47B). Cultures wereincubated for a 24 hour period and conditioned media were harvested andassayed for IL-8 production by ELISA. As shown in FIG. 47A, STF2Δ.EIII+showed potent TLR-5 activity. Regression analysis of the titration curvedetermined the EC₅₀ of batches 2004-044 and 2004-045 to be 1.13 ng/mland 4.34 ng/ml, respectively (Table 1, supra). In both cases, the TLR5specific-activity was at least about 10-fold higher than the controlprotein STF2.OVA. In contrast, 2 preparations of STF2.EIII+ showedsignificantly weaker TLR5 activity than STF2.OVA. The EC₅₀ of STF2.EIII+batches 054 and 057 were about 1195.00 ng/ml and about 197.92 ng/ml.

Antigenicity of STF2.EIII+ and STF2Δ.EIII+

The antigenicity of STF2.EIII+ and STF2Δ.EIII+ was examined by directELISA using a flagellin monoclonal antibody specific for the N-terminalregion of STF2 (6H11, Inotek Pharmaceuticals, Beverly, Mass.) and apanel of WNV-E-specific antibodies (5C5, 5H10, 3A3, 7H2 and 3D9,Bioreliance, Road Rockville, Md.) previously shown to neutralize WestNile virus in vitro. As shown in FIG. 48, a comparison of the reactivityof full length West Nile virus envelope protein with STF2Δ.EIII+revealed that West Nile virus monoclonal antibodies 5C5, 5H10, 3A3 and7H2, but not 3D9 recognize the fusion protein. This pattern ofreactivity is consistent with the proposed location of 5C5, 5H10, 3A3and 7H2 epitopes within EIII. The epitope for 3D9 lies outside of domainIII of the West Nile virus envelope protein. As expected, all West Nilevirus monoclonal antibodies reacted with full length West Nile virusenvelope protein and the flagellin monoclonal only reacted withSTF2Δ.EIII+. Both proteins reacted with a polyclonal West Nile virusenvelope antiserum, but STF2Δ.EIII+ reactivity was somewhat reduced,perhaps due to the reduced number of potential epitopes present in thesmaller domain.

Using 5C5 and 7H10 WNV monoclonal antibodies, a direct antigeniccomparison was made between STF2.EIII+ and STF2Δ.EIII+ (FIGS. 49A, 49B,49C and 49D). In these studies, plates were coated with the indicatedproteins and then detected with polyclonal rabbit anti-E, or mousemonoclonal antibodies as described. As shown in FIGS. 49A, 49B, 49C and49D, both STF2.EIII+ and STF2Δ.EIII+ were readily detected with theflagellin monoclonal antibody with no significant differences inreactivity. However, distinct reactivity with the anti-envelopemonoclonal antibodies was observed. The reactivity of STF2Δ.EIII+ witheither 5C5 or 7H2 was significantly greater than that observed withSTF2.EIII+. Collectively, these results indicate that the flagellin 6H11epitope of STF2Δ.EIII+ is uncompromised and is comparable to theflagellin sequence of STF2.EIII+. They also highlight distinctdifferences in the antigenicity of the EIII domains of these proteinsand indicate that STF2Δ.EIII+ contains more of the critical conformationdependent neutralizing epitopes than STF2.EIII+.

Efficacy and Immunogenicity

Several efficacy studies designed to examine the protective efficacy ourcandidates in C3H/HeN mice following challenge with West Nile virus havebeen completed. Studies typically consisted of 5 groups of mice (10 miceper group) immunized intraperitoneally (i.p.) or subcutaneously (s.c.)on days 0, 14 and 28. On days 21 and 35, sera were harvested and testedfor West Nile virus envelope protein-IgG antibody (ELISA) and theability to neutralize West Nile virus in vitro (PRNT assay). On day 35,mice were challenged with a lethal dose of West Nile virus strain 2741.Survival was monitored for 21 days post-challenge.

Mice were immunized with PBS, Drosophila conditioned medium containingSTF2.E (CM, positive control), 25 μg of STF2Δ.EIII+ i.p., 25 μgSTF2Δ.EIII+ s.c., 25 μg STF2.EIII+ i.p. and 25 μg STF2.EIII+ s.c. TheWest Nile virus envelope protein antibody responses and survival dataare shown FIGS. 50 and 51. By day 35 all groups that receivedSTF2Δ.EIII+ had significant levels of West Nile virus envelope proteinIgG. In contrast, mice that received STF2.EIII+ had no measurable WestNile virus envelope protein antibody response. Administration ofSTF2Δ.EIII+ i.p. or s.c led to 100% survival following West Nile viruschallenge. Consistent with the poor immunogenicity of STF2.EIII+, littleto no protection was provided by this candidate when compared to the PBScontrol. The poor immunogenicity and efficacy of STF2.EIII+ in thisstudy are attributed to the reduced TLR5 activity and/or the weak EIIIepitope reactivity of this protein.

Plaque Reduction Neutralization Titers

To further evaluate the West Nile virus envelope protein antibodyresponse elicited by STF2Δ.EIII+ and potentially correlate protectiveefficacy with neutralizing antibody titers, the plaque reductionneutralization test (PRNT) was performed. Day 35 serum samples fromefficacy studies described above were tested for their ability to blockWest Nile virus infection in cultured Vero cells. Briefly, pooled mouseserum samples were heat-inactivated and serially diluted two-fold in PBSwith 0.5% gelatin. Dilutions starting with 1:10 were incubated withabout 100 pfu of the West Nile virus strain 2741. The virus/serummixture was incubated at about 37° C. for 1 h and then inoculated ontoconfluent monolayers of Vero cells (ATCC, Catalog Number CCL-81,Manassas, Va.) in duplicate wells of 6-well tissue culture plates. Thevirus was allowed to adsorb to the cell monolayer prior to adding a 1%agarose overlay. Infected cell cultures were incubated for 4 days at 37°C. followed by a second agarose overlay containing 4% neutral red. Virusplaques were counted 12 h later. Serum titers that led to 80% reductionin viral plaque numbers (PRNT₈₀) were recorded.

A summary of the PRNT₈₀ data from efficacy studies concerning STF2.EIII+and STF2Δ.EIII+ is presented in Table 2 below. In two independentstudies involving STF2.EIII+ where survival of about 50% or less wasreported, pooled sera failed to inhibit plaque formation. This findingis not surprising given the weak antibody response elicited by thisconstruct. In three efficacy studies involving STF2Δ.EIII+ wheresurvival was about 70% or greater, pooled sera had neutralization titersof 1:40 or better. Neutralization titers of 1:40 or greater typicallycorrelate with protection in vivo.

TABLE 2 Survivial and PRNT₈₀ Results for STF2.EIII+ (SEQ ID NO: 55),STF2Δ.EIII+ (SEQ ID NO: 71) and STF2.E (SEQ ID NO: 159) CM (ControlMedia) Fusion Proteins PRNT₈₀ Batch Candidate Route Study # Survival (%)(dilution) 054 STF2.EIII+ i.p. 3 50 Negative 057 STF2.EIII+ i.p. 4 11Negative 057 STF2.EIII+ s.c. 4 20 negative 044 STF2Δ.EIII+ i.p. 2 701:40 045 STF2Δ.EIII+ i.p. 3 90 1:40 045 STF2Δ.EIII+ s.c. 3 100  1:160045 STF2Δ.EIII+ i.p. 4 100 1:80 045 STF2Δ.EIII+ s.c. 4 100 1:40 — STF2.ECM i.p. 3 90  1:640 — STF2.E CM i.p. 4 —  1:1280STF2Δ.EIIIs+a Modified Version of STF2Δ.EIII+

Protein preparations of STF2Δ.EIII+ tested in the mouse efficacy studiesdescribed above were purified by anion-exchange and size-exclusionchromatography steps carried out under denaturing conditions followed byrefolding using step-wise dialysis. With this process, two predominantspecies that correspond to the monomeric and trimeric forms ofSTF2Δ.EIII+ were generated and present as a mixture in the finalproduct. To minimize the heterogeneity of the final product, newrefolding and purification methods have been developed that favor theproduction of either monomer or trimer. Because it is unclear which formof STF2Δ.EIII+ is the active component or if both are equally potent,both species have been produced in milligram quantities and tested forefficacy in mice.

It was initially unclear as to why STF2Δ.EIII+ refolding resulted in theformation of a trimeric species. However, when the sequence of theSTF2Δ.EIII+ expression construct was re-examined, we identified acysteine residue within the linker sequence that separates STF2Δ fromEIII+. The presence of this cysteine would likely interfere with theformation of the appropriate disulfide bond during refolding and mightaccount for the trimeric form of STF2Δ.EIII+. This unnecessary cysteinewas changed to a serine using site-directed mutagenesis and the modifiedprotein (STF2Δ.EIIIs+) was produced and purified. It should be notedthat refolding the serine-substituted construct yielded only monomericprotein.

Protective efficacy of STF2Δ.EIII+ (monomer) and STF2Δ.EIIIs+(trimer)were evaluated in C3H/HeN mice following challenge with West Nile virus.Five groups of mice (10 per group) were immunized with about 25 ug ofprotein s.c. on days 0, 14 and 28. On days 21 and 35, sera wereharvested and tested for WNV-E IgG antibody (ELISA). On day 38, micewere challenged with a lethal dose of WNV strain 2741 and survival wasmonitored for 21 days. ELISA results from boost 2 (day 35, FIG. 52) andsurvival data (FIG. 53) indicate that all constructs elicitedsignificant levels of WNV-E reactive IgG prior to viral challenge andprovided about 90% to about 100% protection against the lethalinfection. These findings indicate that monomeric or multimeric (e.g.,trimers) forms of STFΔ.EIII+ are efficacious and removal of theadditional cysteine from the construct does not appreciably impactpotency. Removal of the cysteine within the linker sequence may simplifypurification of the protein by reducing heterogeneity following proteinrefolding.

Conclusion

Two recombinant fusion proteins containing the Salmonella typhimuriumflagellin (STF2) fused to EIII+ domain of West Nile virus envelopeprotein have been generated. One includes the full length STF2 sequence(STF2.EIII+) and the other a modified version of STF2 that lacks theinternal hypervariable region of STF2 (STF2Δ.EIII+). Both proteins havebeen expressed in E. coli and purified by conventional means using anionexchange chromatography and gel filtration. STF2.EIII+ was produced as asoluble protein and was purified under non-denaturing conditions. Incontrast, STF2Δ.EIII+ was expressed as an insoluble protein and waspurified under denaturing conditions and refolded by step-wise dialysisto remove urea. In HEK293 IL8 assays, preparations of STF2Δ.EIII+ showedgreater TLR-5 activity than STF2.EIII+.

In envelope protein epitope display analysis using ELISA assays and WestNile virus envelope protein antibodies, STF2Δ.EIII+ displayed more ofthe critical conformation dependent neutralizing epitopes. Consistentwith the potent TLR-5 activity and envelope protein epitope antigenicityobserved with STF2Δ.EIII+, STF2Δ.EIII+ was highly immunogenic andefficacious in mice challenged with a lethal dose of West Nile virus.Because monomeric and trimeric species of STF2Δ.EIII+ were generatedduring the purification process of this protein, a cysteine within thelinker sequence of the expression construct was changed to a serine.Removal of this cysteine eliminated the production of trimeric forms ofthe protein during refolding and resulted in the generation of monomericproduct that displayed potent efficacy in vivo.

Japanese Encephalitis Fusion Protein

JE virus is localized in Asia and northern Australia (about 50,000 caseswith about 10,000 deaths annually). An approved inactivated virusvaccine was recently associated with a case of acute disseminatedencephalomyelitis, prompting the Japanese Ministry of Health, Labor andWelfare to recommend the nationwide suspension of the vaccine. Given thecomplexities of producing inactivated viruses in infected mouse brainsor even in cell culture, and the potential for adverse events associatedwith inactivated viruses, the opportunity for recombinant-based JEvaccine is appealing.

A STF2Δ.JEIII+ fusion construct was constructed. The JE EIII+ DNAfragment was generated synthetically and codon optimized for expressionin E. coli. The sequence was ligated into pET24STF2Δ to generatepETSTF2Δ.JEIII+. Expression constructs have been screened by restrictionanalysis and for expression in E. coli BLR(DE3) by IPTG induction. TheDNA sequence of each construct has been confirmed, and production of theprotein has been scaled up. A batch of material has been generated. Atotal of about 24 mg of material was purified. This material has potentTLR5 activity, acceptable levels of endotoxin (about 0.03 EU/μg) and aA280/A260 ratio of about 1.3.

Flavivirus Peptides

Identification of WNV-E Specific Antibody Epitopes

To identify linear epitopes within the West Nile virus envelope proteinthat are recognized by antisera from STFΔ.EIIIs+ immunized mice, severalsynthetic peptide arrays were generated. One array consisted ofoverlapping peptides of 20 amino acids in length that spanned the entireWest Nile virus domain III and parts of domain II (FIG. 60). ELISAresults with this array identified a highly reactive 20 amino acidsequence that mapped to the N-terminal region of domain III and includedpart of the domain I domain CRVKMEKLQLKGTTYGVCSK (SEQ ID NO: 125). Tofine map this epitope, additional arrays were generated that focused onthe domain I and II junctions (FIGS. 57 and 60). These arrays includedan alanine substitution scan to identify amino acids critical forantibody binding (FIG. 60). As shown in FIGS. 54 and 55, antisera fromSTF2Δ.EIII (monomer and trimer) and STF2Δ.EIIIs+ immunized mice reactedwith peptides that spanned the EI/EIII junction (peptides E-30 to E-42)and included the E2-21 peptide CRVKMEKLQLKGTTYGVCSK (SEQ ID NO: 125).This reactivity was severely reduced when specific amino acids (E6, K7,L10 and K11) were changed to alanines (FIG. 56). Although it is notknown if the antibodies that recognize this epitope are neutralizing,efforts are underway to design and test a peptide vaccine based on thisregion of WNV-E.

Immunogenicity of Pam3Cys.WNV001 Peptide Vaccine

A lipidated West Nile virus envelope protein fused to Pam3Cys on theN-terminal end was synthesized using the 20 amino acid sequenceLTSGHLKCRVKMEKLQLKGT (SEQ ID NO:169) (Putnak, R., et al, Vaccine23:4442-4452 (2005)). The immunogenicity of this peptide was tested inC3H/HeN mice and compared to peptide without Pam3Cys (FIG. 58). Thereactivity of antisera from immunized animals was tested by direct ELISAas described in the legend and the results indicate that thePam3Cys.WNV001 peptide is significantly more immunogenic than thepeptide without the TLR2 modification. The antisera from these studieswill be tested in virus neutralization assays (PRNT) to determine if theantibodies elicited will neutralize West Nile virus in vitro. Thelipidated peptide will also be tested in the West Nile virus challengemodel to assess protective efficacy against a lethal virus challenge.

Assay Development

Competition ELISA Assay Development

To assess the neutralizing potential of antisera derived from immunizedmice, a competition ELISA assay was developed using well-characterizedmonoclonal antibody (7H2) that neutralizes West Nile virus in cultureand reacts with a conformation-sensitive epitope within the EIII domainof the West Nile virus envelope protein antigen. The assay was designedas a capture ELISA that measures the ability of sera from immunizedanimals to prevent 7H2 from binding West Nile virus envelope protein.Serial dilutions ranging from 1:10 to 1:5000 of day 35 mouse antiserafrom efficacy study 4 (FIGS. 50 and 51, Table 2) were incubated withbiotinylated West Nile virus envelope protein and then added to ELISAplates pre-coated with 7H2 monoclonal antibody (Bioreliance, RoadRockville, Md.). Following several washes to remove unbound material,bound West Nile virus envelope protein was detected using avidin-HRP.Results from a representative experiment are shown in FIG. 54. Atdilutions of 1:25, a measurable loss of West Nile virus envelope proteinbinding to 7H2-coated plates was observed when antisera derived fromanimals immunized with STF2Δ.EIIIs where tested. No competition wasdetected with antisera derived from mock immunized animals that receivedPBS in place of antigen. These initial results demonstrate thatantibodies elicited by STF2Δ.EIII+ compete with 7H2 for binding WestNile virus envelope protein. These findings are consistent with theprotection from WNV infection observed in animals immunized withSTF2Δ.EIII+ and help establish a correlation between antibody epitopereactivity in vitro and efficacy in vivo.

Example 2

Materials and Methods

Cloning and Expression of Fusion Proteins

STF2Δ.EIIIs+(SEQ ID NO: 72) and STF2Δ.JEIIIs+(SEQ ID NO: 76) were clonedand expressed as described above.

Protein Purification

Fusion protein (STF2Δ.EIIIs+(SEQ ID NO: 72) was purified as describedabove. The fusion protein STF2Δ.JEIIIs+(SEQ ID NO: 76) was purified asdescribed above for STF2Δ.JEIII+ (SEQ ID NOS: 5, 6). STF2Δ (SEQ ID NO:3) and EIII+ (SEQ ID NO: 7) proteins were purified using conventionalchromatography as described herein and, if expressed in E. coli,required refolding steps due to low solubility in E. coli. Understandard growth and induction conditions described in materials andmethods, STF2Δ (SEQ ID NO: 3) and EIII+ (SEQ ID NO: 7) proteins wereexpressed as insoluble proteins and formed inclusion bodies (IBs). STF2Δ(SEQ ID NO: 3) inclusion bodies were solubilized in 8 M urea in 50 mM NaAcetate, pH 4.0. The solubilized protein was captured on SP fast flowSepharose® under denaturing conditions and selectively eluted with 8 Murea, 50 mM Na Acetate, pH 4.0 buffer containing 0.2 M NaCl. The elutedmaterial was pooled, dialyzed against 50 mM Tris-HCl, pH 8.0, andrefolded by rapid dilution of about 1:10 into 50 mM Tris-HCl, pH 8.0, toa final protein concentration of about 0.1 mg/ml. The refolded SP poolwas loaded directly on Q high performance Sepharose® and bound proteineluted with about 20 column volumes of a linear gradient from 0 to about0.5 M NaCl in 50 mM Tris-HCl, pH 8.0.

EIII+ (SEQ ID NO: 7) inclusion bodies were solubilized with 8 M urea in50 mM Na Acetate, pH 6.3. The protein was applied to SP fast flowSepharose® (GE/Amersham Biosciences). Bound protein was eluted with 50mM Na Acetate, pH 6.3, 8 M urea containing 0.2 M NaCl. SP peak fractionswere pooled and dialyzed against 50 mM Tris-HCl, pH 8.5. To refold theprotein, the dialyzed sample was diluted about 1:10 (final proteinconcentration of about 0.1 mg/ml) in 50 mM Tris-HCl, pH 8.5. Therefolded SP pool was loaded directly on Q high performance Sepharose®(GE/Amersham). Under these conditions, the majority of EIII+ (SEQ ID 7)did not bind Q and eluted with the flow-through fraction. The Q HP FTwas concentrated to about 2 mg/ml (Amicon™ Ultra-15, 5K MW cutoff,Millipore) and applied to size exclusion chromatography (SEC) (SD200,GE/Amersham) pre-equilibrated in Tris-buffered saline (TBS) (25 mMTris-HCl, pH 7.4, 0.13 M NaCl, 2.7 mM KCl).

For use as ELISA reagents, WNV E (SEQ ID NO: 39) and JE E (SEQ ID NO:171) proteins were produced in stable Drosophila Dmel-2 cells with a sixamino acid histidine repeat fused to the c-terminus of the polypeptideaccording to manufacturer's directions (Invitrogen, Carlsbad, Calif.).Stable Drosophila cell pools were expanded as adherent cultures andadapted to suspension growth in selection media (Drosophila SFM, 18 mML-glutamine, 1× penicillin/streptomycin, and 25 μg/mL blasticidin).Protein expression was induced with 0.5 mM CuSO₄ and E protein waspurified by affinity chromatography using nickel NTA according to themanufacturer's directions (Sigma, St. Louis, Mo.).

Efficacy of STF2Δ.JEIIIs+(SEQ ID NO: 76)

Three groups of C57BL/6 mice (20 mice per group) received threeintramuscular (i.m.) immunizations with PBS, 2.5 μg of STF2Δ.JEIIIs+(SEQ ID NO: 76) fusion protein in 1× Tris-buffered saline (TBS) or aboutone-third (⅓) the human dose of JE vaccine (about 0.3 ml ofreconstituted lyophilized killed virus distributed by Sanofi Pasteur,manufactured by BIKEN).

Seven days after each immunization, mice were bled and sera examined byELISA for antibodies to JE E protein. Antigen-specific IgG responses toJE E and STF2 were determined by ELISA. ELISA plates (96 well) (Costar,Catalog No: 9018, Corning, N.Y.) were coated overnight at about 4° C.with about 100 μl/well of recombinant JE E protein expressed inDrosophila and placed in PBS (5 μg/ml). Plates were blocked with 200μl/well of Assay Diluent Buffer (ADB; BD Pharmingen, Catalog No: 555213,San Diego, Calif.) for about one hour at room temperature. The plateswere washed three times in PBS buffer containing 0.05% (v/v) Tween 20(PBS-T). Dilutions of immune sera in ADB were added (about 100 μl/well)and the plates were incubated overnight at about 4° C. The plates werewashed three times with PBS-T. HRP-labeled goat anti-mouse IgGantibodies (Jackson Immunochemical, Catalog No: 115-035-146, West Grove,Pa.) diluted in ADB were added (about 100 μl/well) and the plates wereincubated at room temperature for about 1 hour. The plates were washedthree times with PBS-T. After adding TMB Ultra substrate (Pierce,Catalog No: 34028, Rockford, Ill.) and monitoring color development,A450 was measured on a Tecan Farcyte (Durham, N.C.) microplatespectrophotometer.

Following the third immunization, mice were challenged with the P3 JEstrain (Ni, H., et al., J. Gen Virol. 77:1449-1455 (1996) of JE virusintraperitoneally (i.p.) with an amount of virus equal to ten times thedose needed to cause death in 50% of the mice (10XLD₅₀; one LD₅₀ about10 plaque forming units (pfu).

Results

The immunogenicity of STF2Δ.EIIIs+(SEQ ID NO: 72) was compared with anequimolar amount of STF2Δ (SEQ ID NO: 3) and EIII+ (SEQ ID NO: 7)formulated as a protein cocktail. As shown in FIGS. 81A and 81B,STF2Δ.EIIIs+(SEQ ID NO: 72) elicited measurable WNV-E-specificantibodies, whereas, the STF2Δ (SEQ ID NO: 3)+EIII+ (SEQ ID NO: 7)mixture did not elicit an E-specific response even though flagellinantibodies were readily detectable in these immunized animals. Thispattern of antibody response was also observed following the first boost(days 14) suggesting that a prime and single boost regimen is sufficientto induce a significant antibody response.

Immunizing with EIII+ alone did not elicit E-specific antibodiesdemonstrating the poor immunogencity of this purified antigen asdescribed herein. The efficacy of STF2Δ.EIIIs+(SEQ ID NO: 72) wasdemonstrated by challenging mice with WNV as described herein. As shownin FIG. 82, mice immunized with STF2Δ.EIIIs+(SEQ ID NO: 72) were 100%protected. In contrast, no protective advantage over PBS was observed inmice that received STF2Δ (SEQ ID NO: 3) or EIII⁺ (SEQ ID NO: 7) asseparate immunogens or as a protein mixture. These data show that bothflagellin and EIII+ (SEQ ID NO: 7) are required for protection.

Immunogenicity was also examined in TLR5-deficient mice in a C57BL/6background (Feuillet, V., et al., Proc Natl Acad Sci USA, 103(33):12487-92 (2006)). Mice were immunized as described above (see FIGS. 83Aand 83B), and sera from immunized mice were collected and analyzed forWNV E-specific IgG antibodies. Following immunization withSTF2Δ.EIIIs+(SEQ ID NO: 72), TLR5-deficient animals exhibited markedlylower WNV-E and flagellin IgG responses when compared to wild-type mice(FIGS. 83A and 83B) These studies demonstrate that TLR5 can be requiredto elicit a significant antigen-specific immune response.

Immunogenicity and Efficacy of STF2Δ.JEIIIs+(SEQ ID NO: 76)

The immunogenicity and efficacy of STF2Δ.JEIIIs+ has been demonstratedin mice. To compare the potency of the fusion protein to an approvedvaccine and demonstrate non-inferiority (potency that is equal to orbetter than a vaccine currently in use), an efficacy and immunogenicitystudy was performed using a JE vaccine (JE Vax, distributed by SanofiPasteur, manufactured by BIKEN) approved for use within the US. Threegroups of C57BL/6 mice were immunized three times as described above andsera were collected following each immunization and analyzed for JE Eprotein-specific IgG antibodies. As shown in FIGS. 84A, 84B and 84C,mice immunized with the STF2Δ.JEIIIs+ (SEQ ID NO: 76) fusion proteindeveloped higher JE E protein-specific antibody titers (about 10-fold)than mice immunized with JE Vax (FIGS. 84A, 84B and 84C). These resultssuggest that the fusion protein is more immunogenic with regard to the Eprotein than the JE Vax under these conditions. Once immunized, micewere challenged with a lethal dose of JE virus and the survival results19 days post-challenge are shown (FIG. 85). When challenged with virusdelivered ip, STF2Δ.JEIIIs+(SEQ ID NO: 76) provided comparableprotection (100% efficacy) from a lethal challenge when compared to theJE Vax vaccine. Thus, these data indicate that the fusion proteinsdescribed herein that include JE are not inferior to an approved JEvaccine with regard to efficacy following ip challenge.

Discussion

The presence of a functional TLR5 and the physical association of EIII+(SEQ ID NO: 7) domain to flagellin (STF2Δ (SEQ ID NO: 3)) can generate aprotective immune response. When administered to TLR5 knockout mice as afusion protein (STF2Δ. EIII+), reduced E-specific antibody response wasobserved and when delivered to wild type animals as separate proteincomponents, no E antigen-specific antibody responses were evident. Whenadministered to wild-type mice followed by a challenged with WNV, onlyanimals that received the EIII+ (SEQ ID NO: 7) fused to flagellin (STF2Δ(SEQ ID NO: 3)) survived a lethal West Nile viral dose. In addition,flagellin-JE fusion protein (STF2Δ.JEIIIs+(e.g., SEQ ID NO: 76) similarin design to STF2Δ.EIIIs+ (SEQ ID NO: 72) is both immunogenic andefficacious in mice challenged with a lethal dose of Japaneseencephalitis virus (JEV). Importantly, the efficacy of this recombinantprotein vaccine is not inferior to the approved JE vaccine (JE Vax),which is currently in use within the US and abroad.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A fusion protein comprising a portion of at leastone flagellin and at least a portion of at least one Dengue viralenvelope protein antigen selected from the group consisting of a Den1viral envelope protein antigen, a Den2 viral envelope protein antigen, aDen3 viral envelope protein antigen and a Den4 viral envelope proteinantigen, wherein the portion of the flagellin is a Toll-like Receptor 5agonist, wherein the portion of the Dengue viral envelope protein is anantigen, and wherein the fusion protein activates Toll-like Receptor 5and induces antibodies that neutralize a Dengue virus.
 2. The fusionprotein of claim 1, wherein the flagellin is the Salmonella typhimuriumtype 2 flagellin.
 3. The fusion protein of claim 2, wherein theflagellin includes the amino acid sequence as set forth in SEQ ID NO: 1.4. The fusion protein of claim 1, wherein the Dengue viral envelopeprotein antigen is the Den2 viral envelope protein antigen.
 5. Thefusion protein of claim 4, wherein the Den2 viral envelope proteinantigen includes the amino acid sequence as set forth in SEQ ID NO: 162.6. The fusion protein of claim 1, wherein the Dengue viral envelopeprotein antigen is the Den1 viral envelope protein antigen.
 7. Thefusion protein of claim 1, wherein the Dengue viral envelope proteinantigen is the Den3 viral envelope protein antigen.
 8. The fusionprotein of claim 1, wherein the Dengue viral envelope protein antigen isthe Den4 viral envelope protein antigen.
 9. The fusion protein of claim1, wherein the Dengue viral envelope protein antigen is at least onemember selected from the group consisting of an EIII protein antigen andan EII protein antigen.
 10. The fusion protein of claim 1, wherein theflagellin lacks at least a portion of a hinge region.
 11. The fusionprotein of claim 10, wherein the Dengue viral envelope protein antigenis fused to the flagellin in a the portion of the flagellin that lacksthe hinge region.
 12. The fusion protein of claim 1, wherein theflagellin is fused to a carboxy-terminus of the antigen.
 13. The fusionprotein of claim 1, wherein the flagellin is fused to an amino-terminusof the antigen.
 14. The fusion protein of claim 1, wherein the fusionprotein is a recombinant fusion protein.